Heterodimeric proteins

ABSTRACT

In one aspect, the present invention provides heterodimeric antibodies comprising a first monomer comprising a first heavy chain constant domain comprising a first variant Fc domain and a first antigen binding domain and a second monomer comprising a second heavy chain constant domain comprising a second variant Fc domain and a second antigen binding domain. In an additional aspect the heterodimeric antibody comprises a first monomer comprising a heavy chain comprising a first Fc domain and a single chain Fv region (scFv) that binds a first antigen, wherein the scFv comprises a charged scFv linker. The heterodimeric antibody further comprises a second monomer comprising a first heavy chain comprising a second Fc domain and a first variable heavy chain and a first light chain.

PRIORITY

This application is a continuation-in-part of International PatentApplication No. PCT/US14/11549, filed Jan. 14, 2014, U.S. patentapplication Ser. No. 14/155,334, filed Jan. 14, 2014, 14/205,248, filedMar. 11, 2014 and Ser. No. 14/207,489, filed Mar. 12, 2014. Further,this applications claims the benefit of U.S. Patent Application Nos.61/818,513, filed May 1, 2013, 61/818,344, filed May 1, 2013,61/794,896, filed Mar. 15, 2013, 61/818,401, filed May 1, 2013,61/913,879, filed Dec. 9, 2013, 61/913,832, filed Dec. 9, 2013,61/938,095, filed Feb. 10, 2014 and 61/913,870, filed Dec. 9, 2013, allof which are expressly incorporated by reference in their entirety, andparticularly for all Figures and associated Legends, and for the aminoacid variants disclosed therein.

BACKGROUND OF THE INVENTION

Antibody-based therapeutics have been used successfully to treat avariety of diseases, including cancer and autoimmune/inflammatorydisorders. Yet improvements to this class of drugs are still needed,particularly with respect to enhancing their clinical efficacy. Oneavenue being explored is the engineering of additional and novel antigenbinding sites into antibody-based drugs such that a singleimmunoglobulin molecule co-engages two different antigens. Suchnon-native or alternate antibody formats that engage two differentantigens are often referred to as bispecifics. Because the considerablediversity of the antibody variable region (Fv) makes it possible toproduce an Fv that recognizes virtually any molecule, the typicalapproach to bispecific generation is the introduction of new variableregions into the antibody.

A number of alternate antibody formats have been explored for bispecifictargeting (Chames & Baty, 2009, mAbs 1[6]:1-9; Holliger & Hudson, 2005,Nature Biotechnology 23[9]:1126-1136; Kontermann, mAbs 4(2):182 (2012),all of which are expressly incorporated herein by reference). Initially,bispecific antibodies were made by fusing two cell lines that eachproduced a single monoclonal antibody (Milstein et al., 1983, Nature305:537-540). Although the resulting hybrid hybridoma or quadroma didproduce bispecific antibodies, they were only a minor population, andextensive purification was required to isolate the desired antibody. Anengineering solution to this was the use of antibody fragments to makebispecifics. Because such fragments lack the complex quaternarystructure of a full length antibody, variable light and heavy chains canbe linked in single genetic constructs. Antibody fragments of manydifferent forms have been generated, including diabodies, single chaindiabodies, tandem scFv's, and Fab₂ bispecifics (Chames & Baty, 2009,mAbs 1[6]:1-9; Holliger & Hudson, 2005, Nature Biotechnology23[9]:1126-1136; expressly incorporated herein by reference). Whilethese formats can be expressed at high levels in bacteria and may havefavorable penetration benefits due to their small size, they clearrapidly in vivo and can present manufacturing obstacles related to theirproduction and stability. A principal cause of these drawbacks is thatantibody fragments typically lack the constant region of the antibodywith its associated functional properties, including larger size, highstability, and binding to various Fc receptors and ligands that maintainlong half-life in serum (i.e. the neonatal Fc receptor FcRn) or serve asbinding sites for purification (i.e. protein A and protein G).

More recent work has attempted to address the shortcomings offragment-based bispecifics by engineering dual binding into full lengthantibody-like formats (Wu et al., 2007, Nature Biotechnology25[11]:1290-1297; U.S. Ser. No. 12/477,711; Michaelson et al., 2009,mAbs 1[2]:128-141; PCT/US2008/074693; Zuo et al., 2000, ProteinEngineering 13[5]:361-367; U.S. Ser. No. 09/865,198; Shen et al., 2006,J Biol Chem 281[16]:10706-10714; Lu et al., 2005, Biol Chem280[20]:19665-19672; PCT/US2005/025472; expressly incorporated herein byreference). These formats overcome some of the obstacles of the antibodyfragment bispecifics, principally because they contain an Fc region. Onesignificant drawback of these formats is that, because they build newantigen binding sites on top of the homodimeric constant chains, bindingto the new antigen is always bivalent.

For many antigens that are attractive as co-targets in a therapeuticbispecific format, the desired binding is monovalent rather thanbivalent. For many immune receptors, cellular activation is accomplishedby cross-linking of a monovalent binding interaction. The mechanism ofcross-linking is typically mediated by antibody/antigen immunecomplexes, or via effector cell to target cell engagement. For example,the low affinity Fc gamma receptors (FcγRs) such as FcγRIIa, FcγRIIb,and FcγRIIIa bind monovalently to the antibody Fc region. Monovalentbinding does not activate cells expressing these FcγRs; however, uponimmune complexation or cell-to-cell contact, receptors are cross-linkedand clustered on the cell surface, leading to activation. For receptorsresponsible for mediating cellular killing, for example FcγRIIIa onnatural killer (NK) cells, receptor cross-linking and cellularactivation occurs when the effector cell engages the target cell in ahighly avid format (Bowles & Weiner, 2005, J Immunol Methods 304:88-99,expressly incorporated by reference). Similarly, on B cells theinhibitory receptor FcγRIIb downregulates B cell activation only when itengages into an immune complex with the cell surface B-cell receptor(BCR), a mechanism that is mediated by immune complexation of solubleIgG's with the same antigen that is recognized by the BCR (Heyman 2003,Immunol Lett 88[2]:157-161; Smith and Clatworthy, 2010, Nature ReviewsImmunology 10:328-343; expressly incorporated by reference). As anotherexample, CD3 activation of T-cells occurs only when its associatedT-cell receptor (TCR) engages antigen-loaded MHC on antigen presentingcells in a highly avid cell-to-cell synapse (Kuhns et al., 2006,Immunity 24:133-139). Indeed nonspecific bivalent cross-linking of CD3using an anti-CD3 antibody elicits a cytokine storm and toxicity(Perruche et al., 2009, J Immunol 183 [2]:953-61; Chatenoud & Bluestone,2007, Nature Reviews Immunology 7:622-632; expressly incorporated byreference). Thus for practical clinical use, the preferred mode of CD3co-engagement for redirected killing of targets cells is monovalentbinding that results in activation only upon engagement with theco-engaged target.

Thus while bispecifics generated from antibody fragments sufferbiophysical and pharmacokinetic hurdles, a drawback of those built withfull length antibody-like formats is that they engage co-target antigensmultivalently in the absence of the primary target antigen, leading tononspecific activation and potentially toxicity. The present inventionsolves this problem by introducing a novel set of bispecific formatsthat enable the multivalent co-engagement of distinct target antigens.In addition, the present invention provides novel heterodimerizationvariants that allow for better formation and purification ofheterodimeric proteins, including antibodies.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides heterodimeric antibodiescomprising a first monomer comprising a first heavy chain constantdomain comprising a first variant Fc domain and a first antigen bindingdomain and a second monomer comprising a second heavy chain constantdomain comprising a second variant Fc domain and a second antigenbinding domain.

In an additional aspect the heterodimeric antibody comprises a firstmonomer comprising a heavy chain comprising a first Fc domain and asingle chain Fv region (scFv) that binds a first antigen, wherein thescFv comprises a charged scFv linker. The heterodimeric antibody furthercomprises a second monomer comprising a first heavy chain comprising asecond Fc domain and a first variable heavy chain and a first lightchain. In an additional aspect this charged linker has either a positivecharge from 3 to 8 or a negative charge from 3 to 8 and is selected fromthe group consisting of those linkers depicted in FIG. 9.

In a further aspect, the invention provides heterodimeric antibodycompositions comprising a first monomer comprising a first heavy chainsequence comprising a first variant Fc domain as compared to a human Fcdomain; and a first antigen-binding domain that binds to a firstantigen; and a second heavy chain sequence comprising: a second variantFc domain as compared to a human Fc domain; and a second antigen bindingdomain that binds to a second antigen; wherein the first and secondvariant Fc domains comprise a set of amino acid substitutions selectedfrom the group consisting of the amino acid sets depicted in FIG. 3A-3C.

In an additional aspect, the invention provides heterodimeric antibodycompositions comprising: a first monomer comprising a first heavy chainsequence comprising a first variant Fc domain as compared to a human Fcdomain; and a first antigen-binding domain that binds to a firstantigen; and a second heavy chain sequence comprising a second variantFc domain as compared to a human Fc domain; and a second antigen bindingdomain that binds to CD 19. The second antigen binding domain comprisesa variable heavy chain domain comprising the amino acid sequence ofH1.227 (SEQ ID NO:464) and a variable light chain selected from thegroup consisting of the amino acid sequence of L1.198 (SEQ ID NO:465)and the amino acid sequence of 1.199 (SEQ ID NO:466) as depicted in FIG.21.

In a further aspect, the invention provides a heterodimeric antibodycomposition comprising a first monomer comprising a first heavy chainsequence comprising a first variant Fc domain as compared to a human Fcdomain a first antigen binding domain comprising an anti-CD3 variableregion having a sequence comprising a vhCDR1 having the sequenceT-Y-A-M-Xaa1, wherein Xaa1 is N, S or H (SEQ ID NO:435), a vhCDR2 havingthe sequence R-I-R-S-K-Xaa1-N-Xaa2-Y-A-T-Xaa3-Y-Y-A-Xaa4-S-V-K-G,wherein Xaa1 is Y or A, Xaa2 is N or S, Xaa3 is Y or A and Xaa4 is D orA (SEQ ID NO:436), a vhCDR3 having the sequenceH-G-N-F-G-Xaa1-S-Y-V-S-W-F-Xaa2-Y, wherein Xaa1 is N, D or Q and Xaa2 isA or D (SEQ ID NO:437), a vlCDR1 having the sequenceXaa1-S-S-T-G-A-V-T-Xaa2-Xaa3-Xaa4-Y-A-N, wherein Xaa1 is G, R or K, Xaa2is T or S, Xaa3 is S or G and Xaa4 is N or H, (SEQ ID NO:438), a vlCDR2having the sequence Xaa1-T-N-Xaa2-R-A-Xaa3, wherein Xaa1 is G or D, Xaa2is K or N, and Xaa3 is P or S (SEQ ID NO:439) and a vlCDR3 having thesequence Xaa1-L-W-Y-S-N-Xaa2-W-V, wherein Xaa1 is A or L and Xaa2 is Lor H (SEQ ID NO:440). The heterodimeric antibody further comprises asecond monomer comprising a second heavy chain sequence comprising asecond variant Fc domain as compared to a human Fc domain; and ananti-CD19 antigen binding domain comprising a variable heavy chaindomain comprising the amino acid sequence of H1.227 (SEQ ID NO:464) anda variable light chain selected from the group consisting of the aminoacid sequence of L1.198 (SEQ ID NO:465) and the amino acid sequence of1.199 (SEQ ID NO:466) as depicted in FIG. 21.

In an additional aspect, the invention provides a heterodimeric antibodycomprising a first monomer comprising a heavy chain comprising a firstvariant Fc domain; and a single chain Fv region (scFv) that binds afirst antigen, wherein the scFv comprises a charged scFv linker; and asecond monomer comprising a first heavy chain comprising a secondvariant Fc domain and a first variable heavy chain and the secondmonomer also comprises a first light chain, wherein the first and secondvariant Fc domains comprise amino acid substitution(s) selected from thegroup consisting of those depicted in FIG. 7.

In a further aspect, the invention provides a heterodimeric antibodycomposition comprising a first monomer comprising a first antigenbinding domain comprising an anti-CD3 variable region having a sequencecomprising a vhCDR1 having the sequence T-Y-A-M-Xaa1, wherein Xaa1 is N,S or H (SEQ ID NO:435), a vhCDR2 having the sequenceR-I-R-S-K-Xaa1-N-Xaa2-Y-A-T-Xaa3-Y-Y-A-Xaa4-S-V-K-G, wherein Xaa1 is Yor A, Xaa2 is N or S, Xaa3 is Y or A and Xaa4 is D or A (SEQ ID NO:436),a vhCDR3 having the sequence H-G-N-F-G-Xaa1-S-Y-V-S-W-F-Xaa2-Y, whereinXaa1 is N, D or Q and Xaa2 is A or D (SEQ ID NO:437), a vlCDR1 havingthe sequence Xaa1-S-S-T-G-A-V-T-Xaa2-Xaa3-Xaa4-Y-A-N, wherein Xaa1 is G,R or K, Xaa2 is T or S, Xaa3 is S or G and Xaa4 is N or H, (SEQ IDNO:438), a vlCDR2 having the sequence Xaa1-T-N-Xaa2-R-A-Xaa3, whereinXaa1 is G or D, Xaa2 is K or N, and Xaa3 is P or S (SEQ ID NO:439) and avlCDR3 having the sequence Xaa1-L-W-Y-S-N-Xaa2-W-V, wherein Xaa1 is A orL and Xaa2 is L or H (SEQ ID NO:440). The first monomer also comprises afirst heavy chain sequence comprising a first variant Fc domain ascompared to a human Fc domain. The heterodimeric antibody also comprisesa second monomer comprising a second antigen-binding domain; and asecond heavy chain sequence comprising a second variant Fc domain ascompared to a human Fc domain and wherein the first and second variantFc domains have different amino acid sequences. In some embodiments, theanti-CD3 variable region comprises a vhCDR1 having the sequenceT-Y-A-M-Xaa1, wherein Xaa1 is N, S or H (SEQ ID NO:435), a vhCDR2 havingthe sequence R-I-R-S-K-Xaa1-N-Xaa2-Y-A-T-Xaa3-Y-Y-A-Xaa4-S-V-K-G,wherein Xaa1 is Y or A, Xaa2 is N or S, Xaa3 is Y or A and Xaa4 is D orA (SEQ ID NO:436), a vhCDR3 having the sequenceH-G-N-F-G-Xaa1-S-Y-V-S-W-F-Xaa2-Y, wherein Xaa1 is N, D or Q and Xaa2 isA or D (SEQ ID NO:437), a vlCDR1 having the sequenceXaa1-S-S-T-G-A-V-T-Xaa2-Xaa3-Xaa4-Y-A-N, wherein Xaa1 is G, R or K, Xaa2is T or S, Xaa3 is S or G and Xaa4 is N or H, (SEQ ID NO:438), a vlCDR2having the sequence Xaa1-T-N-Xaa2-R-A-Xaa3, wherein Xaa1 is G or D, Xaa2is K or N, and Xaa3 is P or S (SEQ ID NO:439) and a vlCDR3 having thesequence Xaa1-L-W-Y-S-N-Xaa2-W-V, wherein Xaa1 is A or L and Xaa2 is Lor H (SEQ ID NO:440).

In a further aspect the invention provides heterodimer proteinscomprising a first monomer comprising a first variant heavy chainconstant region and a first fusion partner; and a second monomercomprising a second variant heavy chain constant region and a secondfusion partner, wherein the Fc region of the first and second constantregions comprise a set of amino acid substitutions from FIGS. 3A-3C and12A-12J. IN some cases, the first monomer comprises a third fusionpartner and optionally the second monomer comprises a fourth fusionpartner. The fusion partners are independently selected from the groupconsisting of an immunoglobulin component, a peptide, a cytokine, achemokine, an immune receptor and a blood factor. In some cases, theimmunoglobulin component is selected from the group consisting of Fab,VH, VL, scFv, scFv2, dAb.

In many aspects, one of the first and second variant Fc domains compriseamino acid substitution(s) selected from the group consisting of thosedepicted in FIGS. 6, 7 and/or 12A-12J. In some aspects, the firstantigen binding domain is a scFv covalent attached to the first heavychain constant domain. In additional aspects, the heterodimeric antibodyhas a structure selected from the structures of FIGS. 1B to 1L and 2A to2M. In further aspects, the first and/or second Fc domain of theheterodimeric antibody further comprises amino acid substitution(s)selected from the group consisting of 434A, 434S, 428L, 308F, 259I,428L/434S, 259I/308F, 436I/428L, 436I or V/434S, 436V/428L, 252Y,252Y/254T/256E, 259I/308F/428L, 236A, 239D, 239E, 332E, 332D, 239D/332E,267D, 267E, 328F, 267E/328F, 236A/332E, 239D/332E/330Y, 239D, 332E/330L,236R, 328R, 236R/328R, 236N/267E, 243L, 298A and 299T. In some aspects,one of the first and the second variant Fc domains comprises the aminoacid substitutions 364K/E357Q and the other of comprises the amino acidsubstitutions 368D/370S. These antibodies can further comprise aminoacid substitution(s) selected from the group consisting of those listedin FIG. 7.

In additional aspects the present invention provides nucleic acids,expression vectors and host cells that will produce the heterodimericproteins and antibodies of the invention.

In further aspects the invention provides methods of making theheterodimeric proteins of the invention by culturing host cellscomprising the nucleic acids encoding the heterodimeric proteins andantibodies of the invention under conditions wherein the heterodimer isproduced and recovering the heterodimer.

In a further aspect the invention provides methods of making aheterodimeric antibody of the invention comprising providing a firstnucleic acid encoding a first heavy chain comprising a first heavy chaincomprising a first Fc domain and a single chain Fv region (scFv) thatbinds a first antigen, wherein said scFv comprises a charged linker; andproviding a second nucleic acid encoding a second heavy chain comprisinga second Fc domain a first variable heavy chain; and providing a thirdnucleic acid comprising a light chain. The method additionally comprisesexpressing the first, second and third nucleic acids in a host cell toproduce a first, second and third amino acid sequence, respectively,loading the first, second and third amino acid sequences onto an ionexchange column; and collecting the heterodimeric fraction.

In additional aspects the invention provides methods of treating anindividual in need thereof by administering a heterodimeric antibody orprotein herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Heterodimerization Formats and Variants

FIGS. 1A-1M depict a number of heterodimeric protein formats, includingheterodimeric Fc fusion proteins as well as heterodimeric antibodies.FIG. 1A shows the basic concept of a dimeric Fc region with fourpossible fusion partners A, B, C and D. A, B, C and D are optionally andindependently selected from immunoglobulin domain(s) (e.g. Fab, vH, vL,scFv, scFv₂, scFab, dAb, etc.), peptide(s), cytokines (e.g. IL-2, IL-10,IL-12, GCSF, GM-CSF, etc.), chemokine(s) (e.g. RANTES, CXCL9, CXCL10,CXCL12, etc.), hormone(s) (e.g. FSH, growth hormone), immune receptor(s)(e.g. CTLA-4, TNFR1, TNFRII, other TNFSF, other TNFRSF, etc.) and bloodfactor(s) (e.g. Factor VII, Factor VIII, Factor IX, etc.). Domainsfilled with solid white or solid black are engineered withheterodimerization variants as outlined herein. FIG. 1B depicts the“triple F” format (sometimes also referred to as the “bottle-opener”configuration as discussed below). FIG. 1C shows a “triple F”configuration with another scFv attached to the Fab monomer (this one,along with FIG. 1F, has a greater molecular weight differential aswell). FIG. 1D depicts a “triple F” with another scFv attached to thescFv monomer. FIG. 1E depicts a “three scFv” format. FIG. 1F depicts anadditional Fab attached to the Fab monomer. FIG. 1G depicts a Fab hookedto one of the scFv monomers. FIGS. 1H-1L show additional varieties of“higher multispecificity” embodiments of the “triple F” format, all withone monomer comprising an scFv (and all of which have molecular weightdifferentials which can be exploited for purification of theheterodimers). FIG. 1H shows a “Fab-Fv” format with binding to twodifferent antigens, with FIG. 1I depicting the “Fab-Fv” format withbinding to a single antigen (e.g. bivalent binding to antigen 1). FIGS.1J and 1K depicts a “Fv-Fab” format with similar bivalent or monovalentadditional antigen binding. FIG. 1L depicts one monomer with a CH1-CLattached to the second scFv. FIG. 1M depicts a dual scFv format. In someembodiments the triple F format is not preferred.

FIGS. 2A to 2U depicts a wide variety of the multispecific (e.g.heterodimerization) formats and the combinations of different types ofheterodimerization variants that can be used in the present invention(these are sometimes referred to herein as “heterodimeric scaffolds”).Note in addition that all of these formats can include addition variantsin the Fc region, as more fully discussed below, including “ablation” or“knock out” variants (FIG. 7), Fc variants to alter FcγR binding(FcγRIIb, FcγRIIIa, etc.), Fc variants to alter binding to FcRnreceptor, etc. FIG. 2A shows a dual scFv-Fc format, that, as for allheterodimerization formats herein can include heterodimerizationvariants such as pI variants, knobs in holes (KIH, also referred toherein as steric variants or “skew” variants), charge pairs (a subset ofsteric variants), isosteric variants, and SEED body (“strand-exchangeengineered domain”; see Klein et al., mAbs 4:6 653-663 (2012) and Daviset al, Protein Eng Des Sel 2010 23:195-202) which rely on the fact thatthe CH3 domains of human IgG and IgA do not bind to each other. FIG. 2Bdepicts a bispecific IgG, again with the option of a variety ofheterodimerization variants. FIG. 2C depicts the “one armed” version ofDVD-Ig which utilizes two different variable heavy and variable lightdomains. FIG. 2D is similar, except that rather than an “empty arm”, thevariable heavy and light chains are on opposite heavy chains. FIG. 2E isgenerally referred to as “mAb-Fv”. FIG. 2F depicts a multi-scFv format;as will be appreciated by those in the art, similar to the “A, B, C, D”formats discussed herein, there may be any number of associated scFvs(or, for that matter, any other binding ligands or functionalities).Thus, FIG. 2F could have 1, 2, 3 or 4 scFvs (e.g. for bispecifics, thescFv could be “cis” or “trans”, or both on one “end” of the molecule).FIG. 2G depicts a heterodimeric FabFc with the Fab being formed by twodifferent heavy chains one containing heavy chain Fab sequences and theother containing light chain Fab sequences. FIG. 2H depicts the “onearmed Fab-Fc”, where one heavy chain comprises the Fab. FIG. 2I depictsa “one armed scFv-Fc”, wherein one heavy chain Fc comprises an scFv andthe other heavy chain is “empty”. FIG. 2J shows a scFv-CH3, wherein onlyheavy chain CH3 regions are used, each with their own scFv. FIG. 2Kdepicts a mAb-scFv, wherein one end of the molecule engages an antigenbivalently with a monovalent engagement using an scFv on one of theheavy chains. FIG. 2L depicts the same structure except that both heavychains comprise an additional scFv, which can either bind the sameantigen or different antigens. FIG. 2M shows the “CrossMab” structure,where the problem of multiplex formation due to two different lightchains is addressed by switching sequences in the Fab portion. FIG. 2Ndepicts an scFv, FIG. 2O is a “BiTE” or scFv-scFv linked by a linker asoutlined herein, FIG. 2P depicts a DART, FIG. 2Q depicts a TandAb, andFIG. 2R shows a diabody. FIGS. 2S, 2T and 2U depict additionalalternative scaffold formats that find use in the present invention.

FIG. 3A-3C depicts a number of suitable heterodimerization variants,including skew/steric variants, isosteric variants, pI variants, KIHvariants, etc. for use in the heterodimeric proteins of the invention.As for all the heterodimeric structures herein, each set of theseheterodimerization variants can be combined, optionally andindependently and in any combination in any heterodimeric scaffold. Thevariants at the end of the monomer 1 list are isosteric pI variants,which are generally not use in pairs or sets. In this case, one monomeris engineered to increase or decrease the pI without altering the othermonomer. Thus, although depicted in the “monomer 1” list, these can beincorporated in the appropriate monomer, preserving “strandedness”. Thatis, what is important is that the “strandedness” of the monomer pairsremains intact although variants listed as “monomer 1” variants in thesteric list can be crossed with “monomer 2” variants in the pI list.That is, any set can be combined with any other, regardless of which“monomer” list to which they are associated (as is more fully discussedbelow, in the case where changes in pI are to be used to purify theheterodimeric proteins, the “pI strandedness” is also preserved; forexample, if there are skew variants that happen to alter charge, theyare paired with pI variants on the correct strand; skew variants thatresult in increases in pI are added to the monomer that has increased pIvariants, etc. This is similar to the addition of charged scFv linkers;in that case, as more fully described herein, the correctly charged scFvlinker is added to the correct monomer to preserve the pI difference. Inaddition, each pair of amino acid variants (or where there is a singlemonomer being engineered) can be optionally and independently includedor excluded from any heterodimeric protein, as well as can be optionallyand independently combined.

FIGS. 4A, 4B and 4C depicts a subset of heterodimerization variants ofFIG. 3A-3C finding particular use in the invention.

FIG. 5 depicts a subset of heterodimerization variants of FIG. 3A-3C.

FIG. 6 depicts a list of isotypic and isosteric variant antibodyconstant regions and their respective substitutions. pI_(−) indicateslower pI variants, while pI_(+) indicates higher pI variants. These canbe optionally and independently combined with other heterodimerizationvariants of the invention.

FIG. 7 depicts a number of suitable “knock out” (“KO”) variants toreduce binding to some or all of the FcγR receptors. As is true for manyif not all variants herein, these KO variants can be independently andoptionally combined, both within the set described in FIG. 35 and withany heterodimerization variants outlined herein, including steric and pIvariants. For example, E233P/L234V/L235A/G236del can be combined withany other single or double variant from the list. In addition, while itis preferred in some embodiments that both monomers contain the same KOvariants, it is possible to combine different KO variants on differentmonomers, as well as have only one monomer comprise the KO variant(s).Reference is also made to the Figures and Legends of U.S. Ser. No.61/913,870, all of which is expressly incorporated by reference in itsentirety as it relates to “knock out” or “ablation” variants.

FIG. 8 depicts a number of anti-CD3 scFv engineered disulfides.

FIG. 9 depicts a number of charged scFv linkers that find use inincreasing or decreasing the pI of heterodimeric proteins that utilizeone or more scFv as a component. A single prior art scFv linker with asingle charge is referenced as “Whitlow”, from Whitlow et al., ProteinEngineering 6(8):989-995 (1993). It should be noted that this linker wasused for reducing aggregation and enhancing proteolytic stability inscFvs.

FIGS. 10A and 10B is an additional list of potential heterodimerizationvariants for use in the present invention, including isotypic variants.

FIG. 11 depicts a matrix of possible combinations of heterodimerizationformats, heterodimerization variants (separated into pI variants andsteric variants (which includes charge pair variants), Fc variants, FcRnvariants and combinations. Legend A are suitable FcRn variants: 434A,434S, 428L, 308F, 259I, 428L/434S, 259I/308F, 436I/428L, 436I or V/434S,436V/428L, 252Y, 252Y/254T/256E and 259I/308F/428L. That is, the TripleF format of FIG. 1B can have any of these FcRn variants on either orboth monomer sequences. For clarity, as each heavy chain is different,FcRn variants (as well as the Fc variants) can reside on one or bothmonomers. Legend B are suitable Fc variants: 236A, 239D, 239E, 332E,332D, 239D/332E, 267D, 267E, 328F, 267E/328F, 236A/332E, 239D/332E/330Y,239D, 332E/330L, 236R, 328R, 236R/328R, 236N/267E, 243L, 298A and 299T.(Note, additional suitable Fc variants are found in FIG. 41 of US2006/0024298, the figure and legend of which are hereby incorporated byreference in their entirety). In some cases as described herein, “knockout” or “ablation” variants are used such as depicted in FIG. 7, andthey are included in the definition of Fc variants. As for FcRnvariants, the Fc variants can reside on either strand. Legend C aresuitable pI variants, and these, for brevity are imported from FIGS.3A-3C and 12A-12J, again with the understanding that there is a“strandedness” to pI variants. Legend D are suitable steric variants(including charge pair variants); again, for brevity are imported fromFIGS. 3A-3C and 12A-12J, again with the understanding that there is a“strandedness” to steric variants. Legend E reflects the followingpossible combinations, again, with each variant being independently andoptionally combined from the appropriate source Legend: 1) pI variantsplus FcRn variants; 2) pI variants plus Fc variants; 3) pI variants plusFcRn variants plus Fc variants; 4) steric variants plus FcRn variants;5) steric variants plus Fc variants; 6) steric variants plus FcRnvariants plus Fc variants; 7) pI variants plus steric variants plus FcRnvariants; 8) pI variants plus steric variants plus Fc variants; 9) pIvariants plus steric variants plus FcRn variants plus Fc variants; and10) pI variants plus steric variants. Note any or all of thesecombinations can optionally include or exclude the knock out/ablationvariants in either or both monomers.

FIGS. 12A to 12J depicts additional heterodimerization variant pairs.

Specific Sequences of the Inventions

FIG. 13 depicts the amino acid sequences of wild-type constant regionsused in the invention and the IgG1/G2 fusion.

FIGS. 14A to 14YY depict the amino acid sequences ofstability-optimized, humanized anti-CD3 variant scFvs, variable heavyand variable light sequences. (Note also that the first sequence is thehistidine tagged version for ease of purification). CDRs are underlined.It should be understood that the increased stability of the optimizedvariable and optimized light chains (as well as the scFv chains) can beattributed to framework regions as well as the CDRs. Thus, it should beunderstood that the disclosure of the entire variable region includesthe disclosure of the framework regions, although they are notseparately numbered. In addition, the scFv linkers are shown in grey.Each scFv linker can be replaced with a charged scFv linker as depictedin FIG. 5. That is, any charged scFv linker, whether positive ornegative, including those depicted in FIG. 5, can be substituted for thehighlighted region in FIGS. 14A to 14YY.

FIGS. 15A to 15I depict a collation of all the CD3 vhCDR1-3 and vlCDR1-3sequences useful in the present invention. The sequences of theconsensus CDRs are shown at the end of the Figure.

FIG. 16 shows the sequence of XENP13790, which is XENP12912 (CD3 scFv+disulfide) with the addition of a charged linker.

FIGS. 17A, 17B and 17C. FIG. 17A depicts two different Triple Fembodiments. FIGS. 17B and 17C show the sequences of the Triple Fembodiment of FIG. 17A.

FIG. 18 depicts the sequences of a preferred embodiment of theinvention. The variable regions are underlined, and the charged scFvlinker is in grey.

FIGS. 19A and 19B. The Tm and change in Tm for stability-optimized,humanized anti-CD19 variant scFvs. Amino acid numbering is Kabatnumbering. FIG. 19A as determined by DSF (Differential ScanningFluorimetry) of stability-optimized, humanized anti-CD19 variantscFvs.done at a concentration of 0.2 mg/ml and FIG. 19B was done at 0.4mg/ml.

FIGS. 20A-20K. Amino acid sequences of stability-optimized, humanizedanti-CD19 variant scFvs, variable heavy and variable light sequences.(Note also that the first sequence is the histidine tagged version forease of purification). It should be understood that the increasedstability of the optimized variable and optimized light chains (as wellas the scFv chains) can be attributed to framework regions as well asthe CDRs. Thus, it should be understood that the disclosure of theentire variable region includes the disclosure of the framework regions,although they are not separately numbered.

FIG. 21. Depicts stabilized anti-CD19 Fv regions.

FIGS. 22A and 22B depicts dual-scFv constructs (e.g. as shown in FIG.1M).

FIGS. 23A and 23B depict “bottle opener” constructs (e.g. as shown inFIG. 1B).

FIGS. 24A-24K shows additional sequences of the invention includingisosteric heterodimerization variants.

Data Materials

FIG. 25. Stabilized anti-CD 19 variable regions—competition binding withlabeled anti-CD 19 IgG1 @ 1 μg/mL.

FIG. 26 shows the characterization and comparison of a dual scFv-Fcformat, an anti-CD3/anti-CD19 pair, with the “BiTE” format, using thesame scFvs but no Fc region. As shown, the dual scFv-Fc is less potentthan the BiTE format, but the addition of the Fc region increases thehalf-life in mice by 10 fold.

FIG. 27 depicts that the scFv portions each crossreact with cynomolgusmonkey antigens in an RTCC test. That is, the potency difference betweenthe formats (dual scFv-Fc versus BiTE) translates into cyno monkeys.

FIG. 28 shows that the half life difference also translates into cynomonkeys as between the two formats. The dual scFv-Fc antibody was run atthree different concentrations as shown.

FIG. 29 depicts the projection of pharmacodynamics in monkeys, with theduration of the serum concentration when greater than EC50 is longer forthe dual scFv-Fc format than for BiTE, at 2-3 weeks versus 2-3 days.

FIG. 30 shows the extensive and prolonged B cell killing with the dualscFv-Fc bispecific format. The longer PK of this format enablesprolonged B cell depletion out to 14 days.

FIG. 31 depicts the stability engineering of the anti-CD3/anti-CD19scFv-Fc scFv portions. By identifying and replacing rare amino acids,identifying and replacing amino acids with unusual contacting residues,linker engineering and conversion to VL-VH orientation, substantiallyincreased stability was achieved.

FIG. 32 depicts the improved PK in mice as a result of thestabilization, which resulted in a doubling of the half life in mice forthe anti-CD 19 stabilization.

FIG. 33 shows the production and purification of the “triple F” or“bottle opener” (or as referred to in some of the figures, Fab-scFv-Fc.

FIG. 34 shows the characterization of the anti-CD19/anti-CD3 triple Fformat, which exhibits picomolar cytotoxicity with only monovalentbinding to the target antigens.

FIG. 35 shows the improvement in PK in mice that results from replacingone scFv of a dual scFv-Fc with a Fab. Replacing the anti-CD19 scFv witha Fab doubles the half-life in BL/6 mice from 3 to 6 days.

FIG. 36 depicts the scheme for the “plug and play platform” for thetriple F format. A Fab from any existing mAb can be combined with theanti-CD3 scFv-Fc bispecific format.

FIGS. 37A and 37B depict the characterization of a “plug and play”combination of existing antibodies with the triple F format. FIG. 37Ashows the an anti-CD38 Fab with the anti-CD3 scFv into the triple Fformat, and FIG. 37B shows the Her2/CD3 combination.

FIG. 38 depicts the remarkable “skew” towards heterodimerization usingvariants of the invention. Heterodimerization of over 95% wasaccomplished using one monomer with L368E/K370T and the other with S364Kas compared to the same molecule without the Fc variants.

FIG. 39 shows the B cell depletion in cyno monkey lumph nodes and spleenusing the dual scFv format as compared to the BiTE format.

FIG. 40. List of bevacizumab, Fc-only, and anti-CD19xCD3 heterodimerscontaining isosteric pI substitutions. pI values of each expectedprotein species are indicated.

FIG. 41. Cation exchange chromatography showing purification of theheterodimer species of bevacizumab containing isosteric engineeredconstant regions.

FIG. 42. Cation exchange chromatography showing purification of theheterodimer species of Fc-only variants containing isosteric engineeredconstant regions.

FIG. 43. Cation exchange chromatography showing purification of theheterodimer species of an anti-CD19xCD3 bispecific antibody containingisosteric engineered constant regions. Also shown is an IEF gel ofprotein A purified material as well as the isolated heterodimerbispecific.

FIG. 44. List of bevacizumab and Fc-only variants containing isostericpI substitutions as well as Tm values obtained from DSF.

FIG. 45. List of anti-CD3 and anti-CD19 scFvs containing positively andnegatively charged linkers. Also shown are DSF Tm values.

FIG. 46. Example SEC chromatograms from purified scFvs with positivelycharged linkers.

FIG. 47. Direct binding of anti-CD3 scFvs containing positively chargedlinkers binding to CD4+ T cells (left) or CD20+ cells from PBMCs (tocheck for non-specific binding; right).

FIG. 48. Direct binding of anti-CD3 scFvs containing positively chargedlinkers binding to CD20+ cells from PBMCs (left) or 293E cells (right).

FIG. 49. Example cation exchange purification of XENP13124, which is aFab-scFv-Fc format bispecific antibody targeting CD19 and CD3. Theanti-CD3 scFv contains the positively charged linker (GKPGS)4 to enablepurification.

FIG. 50. Example SEC chromatograms of purified Fab-scFv-Fc formatbispecific antibodies targeting CD 19 and CD3 incubated at variousconcentrations. XENP 13121 (left) contains the standard (GGGGS)4 linkerwhile XENP13124 (right) contains the (GKPGS)4 charged linker. Thecharged linker has the unexpected property of decreasing the amount ofhigh molecular aggregates present.

FIG. 51. RTCC assay with PBMCs and Fab-scFv-Fc format bispecificanti-CD19xCD3 antibodies containing different scFv linkers. Linkers havelittle impact on RTCC activity, except for the highly charged linker(GKGKS)3 which has lower activity.

FIG. 52A to 52O show sequences of the invention that include chargedscFv linkers as well as corresponding controls.

Miscellaneous Other Material

FIG. 53. Literature pIs of the 20 amino acids. It should be noted thatthe listed pIs are calculated as free amino acids; the actual pI of anyside chain in the context of a protein is different, and thus this listis used to show pI trends and not absolute numbers for the purposes ofthe invention.

FIGS. 54A, 54B and 54C. List of all possible reduced pI variants createdfrom isotypic substitutions of IgG1-4. Shown are the pI values for thethree expected species as well as the average delta pI between theheterodimer and the two homodimer species present when the variant heavychain is transfected with IgG1-WT heavy chain.

FIG. 55. List of all possible increased pI variants created fromisotypic substitutions of IgG1-4. Shown are the pI values for the threeexpected species as well as the average delta pI between the heterodimerand the two homodimer species present when the variant heavy chain istransfected with IgG1-WT heavy chain.

FIG. 56 shows the amino acid sequence of the CK and Cλ light constantchains. Residues which contribute to a higher pI (K, R and H) or lowerpI (D and E) are highlighted in bold. Preferred positions formodification to lower the pI are shown in gray. For scaffolds thatcontain one or more light chains, these changes can be used to alter thepI of one or both of the monomers, and can be independently andoptionally combined with all heavy chain variants.

FIGS. 57A to 57E depict the sequences of a number of disulfideconstructs; the first sequence is the scFv construct including theHis(6) tag for convenience of purification, the second sequence is thescFv construct without the tag, the third sequence is the variable heavychain alone and the fourth sequence is the variable light sequencealone. The CDRs are underlined.

DETAILED DESCRIPTION OF THE INVENTION

FIGS. 66 to 80 of WO/2013/055809, the sequences and the accompanyinglegends are incorporated specifically by reference herein. FIGS. 2 to111 and their accompanying legend from U.S. Ser. No. 13/648,951 arespecifically incorporated herein by reference.

I. Overview of Heterodimerization Proteins

The present invention is directed to novel constructs to provideheterodimeric proteins that allow binding to more than one antigen orligand, e.g. to allow for multispecific binding. The heterodimericprotein constructs are based on the self-assembling nature of the two Fcdomains of the heavy chains of antibodies, e.g. two “monomers” thatassemble into a “dimer”. Heterodimeric proteins are made by altering theamino acid sequence of each monomer as more fully discussed below. Thus,the present invention is generally directed to the creation ofheterodimeric proteins including antibodies, which can co-engageantigens in several ways, relying on amino acid variants in the constantregions that are different on each chain to promote heterodimericformation and/or allow for ease of purification of heterodimers over thehomodimers. As discussed more fully below, the heterodimeric proteinscan be antibody variants or based on Fc fusion proteins. In general,heterodimeric antibodies are the focus of the discussion, but as will beappreciated by those in the art and more fully described below, thediscussion applies equally to heterodimeric proteins that

Thus, the present invention provides bispecific antibodies (or, asdiscussed below, trispecific or tetraspecific antibodies can also bemade). An ongoing problem in antibody technologies is the desire for“bispecific” (and/or multispecific) antibodies that bind to two (ormore) different antigens simultaneously, in general thus allowing thedifferent antigens to be brought into proximity and resulting in newfunctionalities and new therapies. In general, these antibodies are madeby including genes for each heavy and light chain into the host cells.This generally results in the formation of the desired heterodimer(A-B), as well as the two homodimers (A-A and B-B). However, a majorobstacle in the formation of multispecific antibodies is the difficultyin purifying the heterodimeric antibodies away from the homodimericantibodies and/or biasing the formation of the heterodimer over theformation of the homodimers.

There are a number of mechanisms that can be used to generate theheterodimers of the present invention. In addition, as will beappreciated by those in the art, these mechanisms can be combined toensure high heterodimerization. Thus, amino acid variants that lead tothe production of heterodimers are referred to as “heterodimerizationvariants”. As discussed below, heterodimerization variants can includesteric variants (e.g. the “knobs and holes” or “skew” variants describedbelow and the “charge pairs” variants described below) as well as “pIvariants”, which allows purification of homodimers away fromheterodimers.

One mechanism is generally referred to in the art as “knobs and holes”(“KIH”) or sometimes herein as “skew” variants, referring to amino acidengineering that creates steric influences to favor heterodimericformation and disfavor homodimeric formation can also optionally beused; this is sometimes referred to as “knobs and holes”; as describedin U.S. Ser. No. 61/596,846 and U.S. Ser. No. 12/875,0015, Ridgway etal., Protein Engineering 9(7):617 (1996); Atwell et al., J. Mol. Biol.1997 270:26; U.S. Pat. No. 8,216,805, US 2012/0149876, all of which arehereby incorporated by reference in their entirety. The Figures identifya number of “monomer A-monomer B” pairs that include “knobs and holes”amino acid substitutions. In addition, as described in Merchant et al.,Nature Biotech. 16:677 (1998), these “knobs and hole” mutations can becombined with disulfide bonds to skew formation to heterodimerization.

An additional mechanism that finds use in the generation of heterodimersis sometimes referred to as “electrostatic steering” or “charge pairs”as described in Gunasekaran et al., J. Biol. Chem. 285(25):19637 (2010),hereby incorporated by reference in its entirety. This is sometimesreferred to herein as “charge pairs”. In this embodiment, electrostaticsare used to skew the formation towards heterodimerization. As those inthe art will appreciate, these may also have an effect on pI, and thuson purification, and thus could in some cases also be considered pIvariants. However, as these were generated to force heterodimerizationand were not used as purification tools, they are classified as “stericvariants”. These include, but are not limited to, D221E/P228E/L368Epaired with D221R/P228R/K409R (e.g. these are “monomer correspondingsets) and C220E/P228E/368E paired with C220R/E224R/P228R/K409R andothers shown in the Figures.

In the present invention, in some embodiments, pI variants are used toalter the pI of one or both of the monomers and thus allowing theisoelectric purification of A-A, A-B and B-B dimeric proteins.

In the present invention, there are several basic mechanisms that canlead to ease of purifying heterodimeric proteins; one relies on the useof pI variants, such that each monomer has a different pI, thus allowingthe isoelectric purification of A-A, A-B and B-B dimeric proteins.Alternatively, some scaffold formats, such as the “triple F” format,also allows separation on the basis of size. As is further outlinedbelow, it is also possible to “skew” the formation of heterodimers overhomodimers. Thus, a combination of steric heterodimerization variantsand pI or charge pair variants find particular use in the invention.Additionally, as more fully outlined below, scaffolds that utilizescFv(s) such as the Triple F format can include charged scFv linkers(either positive or negative), that give a further pI boost forpurification purposes. As will be appreciated by those in the art, someTriple F formats are useful with just charged scFv linkers and noadditional pI adjustments, although the invention does provide the useof skew variants with charged scFv linkers as well (and combinations ofFc, FcRn and KO variants).

In the present invention that utilizes pI as a separation mechanism toallow the purification of heterodimeric proteins, amino acid variantscan be introduced into one or both of the monomer polypeptides; that is,the pI of one of the monomers (referred to herein for simplicity as“monomer A”) can be engineered away from monomer B, or both monomer Aand B change be changed, with the pI of monomer A increasing and the pIof monomer B decreasing. As is outlined more fully below, the pI changesof either or both monomers can be done by removing or adding a chargedresidue (e.g. a neutral amino acid is replaced by a positively ornegatively charged amino acid residue, e.g. glycine to glutamic acid),changing a charged residue from positive or negative to the oppositecharge (aspartic acid to lysine) or changing a charged residue to aneutral residue (e.g. loss of a charge; lysine to serine.). A number ofthese variants are shown in the Figures.

Accordingly, in this embodiment of the present invention provides forcreating a sufficient change in pI in at least one of the monomers suchthat heterodimers can be separated from homodimers. As will beappreciated by those in the art, and as discussed further below, thiscan be done by using a “wild type” heavy chain constant region and avariant region that has been engineered to either increase or decreaseit's pI (wt A−+B or wt A −−B), or by increasing one region anddecreasing the other region (A+−B− or A−B+).

Thus, in general, a component of some embodiments of the presentinvention are amino acid variants in the constant regions of antibodiesthat are directed to altering the isoelectric point (pI) of at leastone, if not both, of the monomers of a dimeric protein to form “pIheterodimers” (when the protein is an antibody, these are referred to as“pI antibodies”) by incorporating amino acid substitutions (“pIvariants” or “pI substitutions”) into one or both of the monomers. Asshown herein, the separation of the heterodimers from the two homodimerscan be accomplished if the pIs of the two monomers differ by as littleas 0.1 pH unit, with 0.2, 0.3, 0.4 and 0.5 or greater all finding use inthe present invention.

As will be appreciated by those in the art, the number of pI variants tobe included on each or both monomer(s) to get good separation willdepend in part on the starting pI of the scFv and Fab of interest. Thatis, to determine which monomer to engineer or in which “direction” (e.g.more positive or more negative), the Fv sequences of the two targetantigens are calculated and a decision is made from there. As is knownin the art, different Fvs will have different starting pIs which areexploited in the present invention. In general, as outlined herein, thepIs are engineered to result in a total pI difference of each monomer ofat least about 0.1 logs, with 0.2 to 0.5 being preferred as outlinedherein.

Furthermore, as will be appreciated by those in the art and outlinedherein, heterodimers can be separated from homodimers on the basis ofsize. For example, as shown in FIGS. 1A-1M and 2A-2U, heterodimers withtwo scFvs can be separated by those of the “triple F” format and abispecific mAb. This can be further exploited in higher valency withadditional antigen binding sites being utilized. For example, asadditionally shown, one monomer will have two Fab fragments and theother will have one scFv, resulting in a differential in size and thusmolecular weight.

In addition, as will be appreciated by those in the art and outlinedherein, the format outlined herein can be expanded to providetrispecific and tetraspecific antibodies as well. In this embodiment,some variations of which are depicted in the FIG. 1A, it will berecognized that it is possible that some antigens are bound divalently(e.g. two antigen binding sites to a single antigen; for example, A andB could be part of a typical bivalent association and C and D can beoptionally present and optionally the same or different). As will beappreciated, any combination of Fab and scFvs can be utilized to achievethe desired result and combinations.

In the case where pI variants are used to achieve heterodimerization, byusing the constant region(s) of the heavy chain(s), a more modularapproach to designing and purifying multispecific proteins, includingantibodies, is provided. Thus, in some embodiments, heterodimerizationvariants (including skew and purification heterodimerization variants)are not included in the variable regions, such that each individualantibody must be engineered. In addition, in some embodiments, thepossibility of immunogenicity resulting from the pI variants issignificantly reduced by importing pI variants from different IgGisotypes such that pI is changed without introducing significantimmunogenicity. Thus, an additional problem to be solved is theelucidation of low pI constant domains with high human sequence content,e.g. the minimization or avoidance of non-human residues at anyparticular position.

A side benefit that can occur with this pI engineering is also theextension of serum half-life and increased FcRn binding. That is, asdescribed in U.S. Ser. No. 13/194,904 (incorporated by reference in itsentirety), lowering the pI of antibody constant domains (including thosefound in antibodies and Fc fusions) can lead to longer serum retentionin vivo. These pI variants for increased serum half life also facilitatepI changes for purification.

In addition, it should be noted that the pI variants of theheterodimerization variants give an additional benefit for the analyticsand quality control process of bispecific antibodies, as, particularlyin the case of CD3 antibodies, the ability to either eliminate, minimizeand distinguish when homodimers are present is significant. Similarly,the ability to reliably test the reproducibility of the heterodimericprotein production is important.

In addition to all or part of a variant heavy constant domain, one orboth of the monomers may contain one or two fusion partners, such thatthe heterodimers form multivalent proteins. As is generally depicted inthe Figures, and specifically FIG. 1A, the fusion partners are depictedas A, B, C and D, with all combinations possible. In general, A, B, Cand D are selected such that the heterodimer is at least bispecific orbivalent in its ability to interact with additional proteins.

As will be appreciated by those in the art and discussed more fullybelow, the heterodimeric fusion proteins of the present invention cantake on a wide variety of configurations, as are generally depicted inFIGS. 1A-1M and 2A-2U. Some figures depict “single ended”configurations, where there is one type of specificity on one “arm” ofthe molecule and a different specificity on the other “arm”. Otherfigures depict “dual ended” configurations, where there is at least onetype of specificity at the “top” of the molecule and one or moredifferent specificities at the “bottom” of the molecule. Furthermore asis shown, these two configurations can be combined, where there can betriple or quadruple specificities based on the particular combination.Thus, the present invention provides “multispecific” binding proteins,including multispecific antibodies. Thus, the present invention isdirected to novel immunoglobulin compositions that co-engage at least afirst and a second antigen. First and second antigens of the inventionare herein referred to as antigen-1 and antigen-2 respectively.

One heterodimeric scaffold that finds particular use in the presentinvention is the “triple F” or “bottle opener” scaffold format. In thisembodiment, one heavy chain of the antibody contains an single chain Fv(“scFv”, as defined below) and the other heavy chain is a “regular” FAbformat, comprising a variable heavy chain and a light chain. Thisstructure is sometimes referred to herein as “triple F” format(scFv-FAb-Fc) or the “bottle-opener” format, due to a rough visualsimilarity to a bottle-opener (see FIG. 1B). The two chains are broughttogether by the use of amino acid variants in the constant regions (e.g.the Fc domain and/or the hinge region) that promote the formation ofheterodimeric antibodies as is described more fully below.

There are several distinct advantages to the present “triple F” format.As is known in the art, antibody analogs relying on two scFv constructsoften have stability and aggregation problems, which can be alleviatedin the present invention by the addition of a “regular” heavy and lightchain pairing. In addition, as opposed to formats that rely on two heavychains and two light chains, there is no issue with the incorrectpairing of heavy and light chains (e.g. heavy 1 pairing with light 2,etc.)

In addition to all or part of a variant heavy constant domain, one orboth of the monomers may contain one or two fusion partners, such thatthe heterodimers form multivalent proteins. As is generally depicted inthe FIG. 64 of U.S. Ser. No. 13/648,951, hereby incorporated byreference with its accompanying legend, the fusion partners are depictedas A, B, C and D, with all combinations possible. In general, A, B, Cand D are selected such that the heterodimer is at least bispecific orbivalent in its ability to interact with additional proteins. In thecontext of the present “triple F” format, generally A and B are an scFvand a Fv (as will be appreciated, either monomer can contain the scFvand the other the Fv/Fab) and then optionally one or two additionalfusion partners.

Furthermore, as outlined herein, additional amino acid variants may beintroduced into the bispecific antibodies of the invention, to addadditional functionalities. For example, amino acid changes within theFc region can be added (either to one monomer or both) to facilitateincreased ADCC or CDC (e.g. altered binding to Fcγ receptors); to allowor increase yield of the addition of toxins and drugs (e.g. for ADC), aswell as to increase binding to FcRn and/or increase serum half-life ofthe resulting molecules. As is further described herein and as will beappreciated by those in the art, any and all of the variants outlinedherein can be optionally and independently combined with other variants.

Similarly, another category of functional variants are “Fcγ ablationvariants” or “Fc knock out (FcKO or KO) variants. In these embodiments,for some therapeutic applications, it is desirable to reduce or removethe normal binding of the Fc domain to one or more or all of the Fcγreceptors (e.g. FcγR1, FcγRIIa, FcγRIIb, FcγRIIIa, etc.) to avoidadditional mechanisms of action. That is, for example, in manyembodiments, particularly in the use of bispecific antibodies that bindCD3 monovalently and a tumor antigen on the other (e.g. CD 19, her2/neu,etc.), it is generally desirable to ablate FcγRIIIa binding to eliminateor significantly reduce ADCC activity.

DEFINITIONS

In order that the application may be more completely understood, severaldefinitions are set forth below. Such definitions are meant to encompassgrammatical equivalents.

By “ablation” herein is meant a decrease or removal of activity. Thusfor example, “ablating FcγR binding” means the Fc region amino acidvariant has less than 50% starting binding as compared to an Fc regionnot containing the specific variant, with less than 70-80-90-95-98% lossof activity being preferred, and in general, with the activity beingbelow the level of detectable binding in a Biacore assay. Of particularuse in the ablation of FcγR binding are those shown in FIG. 7.

By “ADCC” or “antibody dependent cell-mediated cytotoxicity” as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell. ADCC is correlated withbinding to FcγRIIIa; increased binding to FcγRIIIa leads to an increasein ADCC activity.

By “ADCP” or antibody dependent cell-mediated phagocytosis as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause phagocytosis of the target cell.

By “modification” herein is meant an amino acid substitution, insertion,and/or deletion in a polypeptide sequence or an alteration to a moietychemically linked to a protein. For example, a modification may be analtered carbohydrate or PEG structure attached to a protein. By “aminoacid modification” herein is meant an amino acid substitution,insertion, and/or deletion in a polypeptide sequence. For clarity,unless otherwise noted, the amino acid modification is always to anamino acid coded for by DNA, e.g. the 20 amino acids that have codons inDNA and RNA.

By “amino acid substitution” or “substitution” herein is meant thereplacement of an amino acid at a particular position in a parentpolypeptide sequence with a different amino acid. In particular, in someembodiments, the substitution is to an amino acid that is not naturallyoccurring at the particular position, either not naturally occurringwithin the organism or in any organism. For example, the substitutionE272Y refers to a variant polypeptide, in this case an Fc variant, inwhich the glutamic acid at position 272 is replaced with tyrosine. Forclarity, a protein which has been engineered to change the nucleic acidcoding sequence but not change the starting amino acid (for exampleexchanging CGG (encoding arginine) to CGA (still encoding arginine) toincrease host organism expression levels) is not an “amino acidsubstitution”; that is, despite the creation of a new gene encoding thesame protein, if the protein has the same amino acid at the particularposition that it started with, it is not an amino acid substitution.

By “amino acid insertion” or “insertion” as used herein is meant theaddition of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, -233E or 233E designates an insertionof glutamic acid after position 233 and before position 234.Additionally, -233ADE or A233ADE designates an insertion of AlaAspGluafter position 233 and before position 234.

By “amino acid deletion” or “deletion” as used herein is meant theremoval of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, E233- or E233# or E233( ) designatesa deletion of glutamic acid at position 233. Additionally, EDA233- orEDA233# designates a deletion of the sequence GluAspAla that begins atposition 233.

By “variant protein” or “protein variant”, or “variant” as used hereinis meant a protein that differs from that of a parent protein by virtueof at least one amino acid modification. Protein variant may refer tothe protein itself, a composition comprising the protein, or the aminosequence that encodes it. Preferably, the protein variant has at leastone amino acid modification compared to the parent protein, e.g. fromabout one to about seventy amino acid modifications, and preferably fromabout one to about five amino acid modifications compared to the parent.As described below, in some embodiments the parent polypeptide, forexample an Fc parent polypeptide, is a human wild type sequence, such asthe Fc region from IgG1, IgG2, IgG3 or IgG4, although human sequenceswith variants can also serve as “parent polypeptides”, for example theIgG1/2 hybrid of FIG. 13. The protein variant sequence herein willpreferably possess at least about 80% identity with a parent proteinsequence, and most preferably at least about 90% identity, morepreferably at least about 95-98-99% identity. Variant protein can referto the variant protein itself, compositions comprising the proteinvariant, or the DNA sequence that encodes it. Accordingly, by “antibodyvariant” or “variant antibody” as used herein is meant an antibody thatdiffers from a parent antibody by virtue of at least one amino acidmodification, “IgG variant” or “variant IgG” as used herein is meant anantibody that differs from a parent IgG (again, in many cases, from ahuman IgG sequence) by virtue of at least one amino acid modification,and “immunoglobulin variant” or “variant immunoglobulin” as used hereinis meant an immunoglobulin sequence that differs from that of a parentimmunoglobulin sequence by virtue of at least one amino acidmodification. “Fc variant” or “variant Fc” as used herein is meant aprotein comprising an amino acid modification in an Fc domain. The Fcvariants of the present invention are defined according to the aminoacid modifications that compose them. Thus, for example, N434S or 434Sis an Fc variant with the substitution serine at position 434 relativeto the parent Fc polypeptide, wherein the numbering is according to theEU index. Likewise, M428L/N434S defines an Fc variant with thesubstitutions M428L and N434S relative to the parent Fc polypeptide. Theidentity of the WT amino acid may be unspecified, in which case theaforementioned variant is referred to as 428L/434S. It is noted that theorder in which substitutions are provided is arbitrary, that is to saythat, for example, 428L/434S is the same Fc variant as M428L/N434S, andso on. For all positions discussed in the present invention that relateto antibodies, unless otherwise noted, amino acid position numbering isaccording to the EU index. The EU index or EU index as in Kabat or EUnumbering scheme refers to the numbering of the EU antibody (Edelman etal., 1969, Proc Natl Acad Sci USA 63:78-85, hereby entirely incorporatedby reference.) The modification can be an addition, deletion, orsubstitution. Substitutions can include naturally occurring amino acidsand, in some cases, synthetic amino acids. Examples include U.S. Pat.No. 6,586,207; WO 98/48032; WO 03/073238; US2004-0214988A1; WO05/35727A2; WO 05/74524A2; J. W. Chin et al., (2002), Journal of theAmerican Chemical Society 124:9026-9027; J. W. Chin, & P. G. Schultz,(2002), ChemBioChem 11:1135-1137; J. W. Chin, et al., (2002), PICASUnited States of America 99:11020-11024; and, L. Wang, & P. G. Schultz,(2002), Chem. 1-10, all entirely incorporated by reference.

As used herein, “protein” herein is meant at least two covalentlyattached amino acids, which includes proteins, polypeptides,oligopeptides and peptides. The peptidyl group may comprise naturallyoccurring amino acids and peptide bonds, or synthetic peptidomimeticstructures, i.e. “analogs”, such as peptoids (see Simon et al., PNAS USA89(20):9367 (1992), entirely incorporated by reference). The amino acidsmay either be naturally occurring or synthetic (e.g. not an amino acidthat is coded for by DNA); as will be appreciated by those in the art.For example, homo-phenylalanine, citrulline, ornithine and noreleucineare considered synthetic amino acids for the purposes of the invention,and both D- and L-(R or S) configured amino acids may be utilized. Thevariants of the present invention may comprise modifications thatinclude the use of synthetic amino acids incorporated using, forexample, the technologies developed by Schultz and colleagues, includingbut not limited to methods described by Cropp & Shultz, 2004, TrendsGenet. 20(12):625-30, Anderson et al., 2004, Proc Natl Acad Sci USA 101(2):7566-71, Zhang et al., 2003, 303(5656):371-3, and Chin et al., 2003,Science 301(5635):964-7, all entirely incorporated by reference. Inaddition, polypeptides may include synthetic derivatization of one ormore side chains or termini, glycosylation, PEGylation, circularpermutation, cyclization, linkers to other molecules, fusion to proteinsor protein domains, and addition of peptide tags or labels.

By “residue” as used herein is meant a position in a protein and itsassociated amino acid identity. For example, Asparagine 297 (alsoreferred to as Asn297 or N297) is a residue at position 297 in the humanantibody IgG1.

By “Fab” or “Fab region” as used herein is meant the polypeptide thatcomprises the VH, CH1, VL, and CL immunoglobulin domains. Fab may referto this region in isolation, or this region in the context of a fulllength antibody, antibody fragment or Fab fusion protein. By “Fv” or “Fvfragment” or “Fv region” as used herein is meant a polypeptide thatcomprises the VL and VH domains of a single antibody.

By “IgG subclass modification” or “isotype modification” as used hereinis meant an amino acid modification that converts one amino acid of oneIgG isotype to the corresponding amino acid in a different, aligned IgGisotype. For example, because IgG1 comprises a tyrosine and IgG2 aphenylalanine at EU position 296, a F296Y substitution in IgG2 isconsidered an IgG subclass modification.

By “non-naturally occurring modification” as used herein is meant anamino acid modification that is not isotypic. For example, because noneof the IgGs comprise a serine at position 434, the substitution 434S inIgG1, IgG2, IgG3, or IgG4 (or hybrids thereof) is considered anon-naturally occurring modification.

By “amino acid” and “amino acid identity” as used herein is meant one ofthe naturally occurring amino acids that are coded for by DNA and RNA.

By “effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include but are not limited toADCC, ADCP, and CDC.

By “IgG Fc ligand” as used herein is meant a molecule, preferably apolypeptide, from any organism that binds to the Fc region of an IgGantibody to form an Fc/Fc ligand complex. Fc ligands include but are notlimited to FcγRIs, FcγRIIs, FcγRIIIs, FcRn, C1q, C3, mannan bindinglectin, mannose receptor, staphylococcal protein A, streptococcalprotein G, and viral FcγR. Fc ligands also include Fc receptor homologs(FcRH), which are a family of Fc receptors that are homologous to theFcγRs (Davis et al., 2002, Immunological Reviews 190:123-136, entirelyincorporated by reference). Fc ligands may include undiscoveredmolecules that bind Fc. Particular IgG Fc ligands are FcRn and Fc gammareceptors. By “Fc ligand” as used herein is meant a molecule, preferablya polypeptide, from any organism that binds to the Fc region of anantibody to form an Fc/Fc ligand complex.

By “Fc gamma receptor”, “FcγR” or “FcqammaR” as used herein is meant anymember of the family of proteins that bind the IgG antibody Fc regionand is encoded by an FcγR gene. In humans this family includes but isnot limited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb, andFcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotypesH131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), andFcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (includingallotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIb-NA1and FcγRIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, entirelyincorporated by reference), as well as any undiscovered human FcγRs orFcγR isoforms or allotypes. An FcγR may be from any organism, includingbut not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγRsinclude but are not limited to FcγRI (CD64), FcγRII (CD32), FcγRIII(CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγRsor FcγR isoforms or allotypes.

By “FcRn” or “neonatal Fc Receptor” as used herein is meant a proteinthat binds the IgG antibody Fc region and is encoded at least in part byan FcRn gene. The FcRn may be from any organism, including but notlimited to humans, mice, rats, rabbits, and monkeys. As is known in theart, the functional FcRn protein comprises two polypeptides, oftenreferred to as the heavy chain and light chain. The light chain isbeta-2-microglobulin and the heavy chain is encoded by the FcRn gene.Unless otherwise noted herein, FcRn or an FcRn protein refers to thecomplex of FcRn heavy chain with beta-2-microglobulin. A variety of FcRnvariants used to increase binding to the FcRn receptor, and in somecases, to increase serum half-life, are shown in the Figure Legend ofFIG. 11.

By “parent polypeptide” as used herein is meant a starting polypeptidethat is subsequently modified to generate a variant. The parentpolypeptide may be a naturally occurring polypeptide, or a variant orengineered version of a naturally occurring polypeptide. Parentpolypeptide may refer to the polypeptide itself, compositions thatcomprise the parent polypeptide, or the amino acid sequence that encodesit. Accordingly, by “parent immunoglobulin” as used herein is meant anunmodified immunoglobulin polypeptide that is modified to generate avariant, and by “parent antibody” as used herein is meant an unmodifiedantibody that is modified to generate a variant antibody. It should benoted that “parent antibody” includes known commercial, recombinantlyproduced antibodies as outlined below.

By “Fc fusion protein” or “immunoadhesin” herein is meant a proteincomprising an Fc region, generally linked (optionally through a linkermoiety, as described herein) to a different protein, such as a bindingmoiety to a target protein, as described herein. In some cases, onemonomer of the heterodimeric protein comprises an antibody heavy chain(either including an scFv or further including a light chain) and theother monomer is a Fc fusion, comprising a variant Fc domain and aligand. In some embodiments, these “half antibody-half fusion proteins”are referred to as “Fusionbodies”.

By “position” as used herein is meant a location in the sequence of aprotein. Positions may be numbered sequentially, or according to anestablished format, for example the EU index for antibody numbering.

By “target antigen” as used herein is meant the molecule that is boundspecifically by the variable region of a given antibody. A targetantigen may be a protein, carbohydrate, lipid, or other chemicalcompound. A wide number of suitable target antigens are described below.

By “strandedness” in the context of the monomers of the heterodimericproteins of the invention herein is meant that, similar to the twostrands of DNA that “match”, heterodimerization variants areincorporated into each monomer so as to preserve the ability to “match”to form heterodimers. For example, if some pI variants are engineeredinto monomer A (e.g. making the pI higher) then steric variants that are“charge pairs” that can be utilized as well do not interfere with the pIvariants, e.g. the charge variants that make a pI higher are put on thesame “strand” or “monomer” to preserve both functionalities.

By “target cell” as used herein is meant a cell that expresses a targetantigen.

By “variable region” as used herein is meant the region of animmunoglobulin that comprises one or more Ig domains substantiallyencoded by any of the V.kappa., V.lamda., and/or VH genes that make upthe kappa, lambda, and heavy chain immunoglobulin genetic locirespectively.

By “wild type or WT” herein is meant an amino acid sequence or anucleotide sequence that is found in nature, including allelicvariations. A WT protein has an amino acid sequence or a nucleotidesequence that has not been intentionally modified.

The antibodies of the present invention are generally isolated orrecombinant. “Isolated,” when used to describe the various polypeptidesdisclosed herein, means a polypeptide that has been identified andseparated and/or recovered from a cell or cell culture from which it wasexpressed. Ordinarily, an isolated polypeptide will be prepared by atleast one purification step. An “isolated antibody,” refers to anantibody which is substantially free of other antibodies havingdifferent antigenic specificities.

“Specific binding” or “specifically binds to” or is “specific for” aparticular antigen or an epitope means binding that is measurablydifferent from a non-specific interaction. Specific binding can bemeasured, for example, by determining binding of a molecule compared tobinding of a control molecule, which generally is a molecule of similarstructure that does not have binding activity. For example, specificbinding can be determined by competition with a control molecule that issimilar to the target.

Specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KD for an antigen orepitope of at least about 10-4 M, at least about 10-5 M, at least about10-6 M, at least about 10-7 M, at least about 10-8 M, at least about10-9 M, alternatively at least about 10-10 M, at least about 10-11 M, atleast about 10-12 M, or greater, where KD refers to a dissociation rateof a particular antibody-antigen interaction. Typically, an antibodythat specifically binds an antigen will have a KD that is 20-, 50-,100-, 500-, 1000-, 5,000-, 10,000- or more times greater for a controlmolecule relative to the antigen or epitope.

Also, specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KA or Ka for an antigenor epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- ormore times greater for the epitope relative to a control, where KA or Karefers to an association rate of a particular antibody-antigeninteraction.

Heterodimeric Proteins

The present invention is directed to the generation of multispecific,particularly bispecific binding proteins, and in particular,multispecific antibodies. The present invention generally relies on theuse of engineered or variant Fc domains that can self-assemble inproduction cells to produce heterodimeric proteins, and methods togenerate and purify such heterodimeric proteins.

Antibodies

The present invention relates to the generation of multispecificantibodies, generally therapeutic antibodies. As is discussed below, theterm “antibody” is used generally. Antibodies that find use in thepresent invention can take on a number of formats as described herein,including traditional antibodies as well as antibody derivatives,fragments and mimetics, described below. In general, the term “antibody”includes any polypeptide that includes at least one constant domain,including, but not limited to, CH1 CH2, CH3 and CL.

Traditional antibody structural units typically comprise a tetramer.Each tetramer is typically composed of two identical pairs ofpolypeptide chains, each pair having one “light” (typically having amolecular weight of about 25 kDa) and one “heavy” chain (typicallyhaving a molecular weight of about 50-70 kDa). Human light chains areclassified as kappa and lambda light chains. The present invention isdirected to the IgG class, which has several subclasses, including, butnot limited to IgG1, IgG2, IgG3, and IgG4. Thus, “isotype” as usedherein is meant any of the subclasses of immunoglobulins defined by thechemical and antigenic characteristics of their constant regions. Itshould be understood that therapeutic antibodies can also comprisehybrids of isotypes and/or subclasses. For example, as shown in USPublication 2009/0163699, incorporated by reference, the presentinvention covers pI engineering of IgG1/G2 hybrids.

The amino-terminal portion of each chain includes a variable region ofabout 100 to 110 or more amino acids primarily responsible for antigenrecognition, generally referred to in the art and herein as the “Fvdomain” or “Fv region”. In the variable region, three loops are gatheredfor each of the V domains of the heavy chain and light chain to form anantigen-binding site. Each of the loops is referred to as acomplementarity-determining region (hereinafter referred to as a “CDR”),in which the variation in the amino acid sequence is most significant.“Variable” refers to the fact that certain segments of the variableregion differ extensively in sequence among antibodies. Variabilitywithin the variable region is not evenly distributed. Instead, the Vregions consist of relatively invariant stretches called frameworkregions (FRs) of 15-30 amino acids separated by shorter regions ofextreme variability called “hypervariable regions” that are each 9-15amino acids long or longer.

Each VH and VL is composed of three hypervariable regions(“complementary determining regions,” “CDRs”) and four FRs, arrangedfrom amino-terminus to carboxy-terminus in the following order:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

The hypervariable region generally encompasses amino acid residues fromabout amino acid residues 24-34 (LCDR1; “L” denotes light chain), 50-56(LCDR2) and 89-97 (LCDR3) in the light chain variable region and aroundabout 31-35B (HCDR1; “H” denotes heavy chain), 50-65 (HCDR2), and 95-102(HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OFPROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991) and/or thoseresidues forming a hypervariable loop (e.g. residues 26-32 (LCDR1),50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chainvariable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917.Specific CDRs of the invention are described below.

Throughout the present specification, the Kabat numbering system isgenerally used when referring to a residue in the variable domain(approximately, residues 1-107 of the light chain variable region andresidues 1-113 of the heavy chain variable region) and the EU numberingsystem for Fc regions (e.g., Kabat et al., supra (1991)).

The CDRs contribute to the formation of the antigen-binding, or morespecifically, epitope binding site of antibodies. “Epitope” refers to adeterminant that interacts with a specific antigen binding site in thevariable region of an antibody molecule known as a paratope. Epitopesare groupings of molecules such as amino acids or sugar side chains andusually have specific structural characteristics, as well as specificcharge characteristics. A single antigen may have more than one epitope.

The epitope may comprise amino acid residues directly involved in thebinding (also called immunodominant component of the epitope) and otheramino acid residues, which are not directly involved in the binding,such as amino acid residues which are effectively blocked by thespecifically antigen binding peptide; in other words, the amino acidresidue is within the footprint of the specifically antigen bindingpeptide.

Epitopes may be either conformational or linear. A conformationalepitope is produced by spatially juxtaposed amino acids from differentsegments of the linear polypeptide chain. A linear epitope is oneproduced by adjacent amino acid residues in a polypeptide chain.Conformational and nonconformational epitopes may be distinguished inthat the binding to the former but not the latter is lost in thepresence of denaturing solvents.

An epitope typically includes at least 3, and more usually, at least 5or 8-10 amino acids in a unique spatial conformation. Antibodies thatrecognize the same epitope can be verified in a simple immunoassayshowing the ability of one antibody to block the binding of anotherantibody to a target antigen, for example “binning.”

The carboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function. Kabat et al. collectednumerous primary sequences of the variable regions of heavy chains andlight chains. Based on the degree of conservation of the sequences, theyclassified individual primary sequences into the CDR and the frameworkand made a list thereof (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5thedition, NIH publication, No. 91-3242, E. A. Kabat et al., entirelyincorporated by reference).

In the IgG subclass of immunoglobulins, there are several immunoglobulindomains in the heavy chain. By “immunoglobulin (Ig) domain” herein ismeant a region of an immunoglobulin having a distinct tertiarystructure. Of interest in the present invention are the heavy chaindomains, including, the constant heavy (CH) domains and the hingedomains. In the context of IgG antibodies, the IgG isotypes each havethree CH regions. Accordingly, “CH” domains in the context of IgG are asfollows: “CH1” refers to positions 118-220 according to the EU index asin Kabat. “CH2” refers to positions 237-340 according to the EU index asin Kabat, and “CH3” refers to positions 341-447 according to the EUindex as in Kabat. As shown herein and described below, the pI variantscan be in one or more of the CH regions, as well as the hinge region,discussed below.

It should be noted that the sequences depicted herein start at the CH1region, position 118; the variable regions are not included except asnoted. For example, the first amino acid of SEQ ID NO: 2, whiledesignated as position“1” in the sequence listing, corresponds toposition 118 of the CH1 region, according to EU numbering.

Another type of Ig domain of the heavy chain is the hinge region. By“hinge” or “hinge region” or “antibody hinge region” or “immunoglobulinhinge region” herein is meant the flexible polypeptide comprising theamino acids between the first and second constant domains of anantibody. Structurally, the IgG CH1 domain ends at EU position 220, andthe IgG CH2 domain begins at residue EU position 237. Thus for IgG theantibody hinge is herein defined to include positions 221 (D221 in IgG1)to 236 (G236 in IgG1), wherein the numbering is according to the EUindex as in Kabat. In some embodiments, for example in the context of anFc region, the lower hinge is included, with the “lower hinge” generallyreferring to positions 226 or 230. As noted herein, pI variants can bemade in the hinge region as well.

The light chain generally comprises two domains, the variable lightdomain (containing the light chain CDRs and together with the variableheavy domains forming the Fv region), and a constant light chain region(often referred to as CL or Cκ.

Another region of interest for additional substitutions, outlined below,is the Fc region. By “Fc” or “Fc region” or “Fc domain” as used hereinis meant the polypeptide comprising the constant region of an antibodyexcluding the first constant region immunoglobulin domain and in somecases, part of the hinge. Thus Fc refers to the last two constant regionimmunoglobulin domains of IgA, IgD, and IgG, the last three constantregion immunoglobulin domains of IgE and IgM, and the flexible hingeN-terminal to these domains. For IgA and IgM, Fc may include the Jchain. For IgG, the Fc domain comprises immunoglobulin domains Cγ2 andCγ3 (Cγ2 and Cγ3) and the lower hinge region between Cγ1 (Cγ1) and Cγ2(Cγ2). Although the boundaries of the Fc region may vary, the human IgGheavy chain Fc region is usually defined to include residues C226 orP230 to its carboxyl-terminus, wherein the numbering is according to theEU index as in Kabat. In some embodiments, as is more fully describedbelow, amino acid modifications are made to the Fc region, for exampleto alter binding to one or more FcγR receptors or to the FcRn receptor.

Accordingly, in some embodiments the present invention providesheterodimeric antibodies that rely on the use of two different heavychain variant Fc domains that will self-assemble to form heterodimericantibodies.

In some embodiments, the antibodies are full length. By “full lengthantibody” herein is meant the structure that constitutes the naturalbiological form of an antibody, including variable and constant regions,including one or more modifications as outlined herein, particularly inthe Fc domains to allow either heterodimerization formation or thepurification of heterodimers away from homodimers. A full lengthheterodimeric antibody is two heavy chains with different Fc domains andeither two light chains or a common light chain.

Alternatively, the antibodies can include a variety of structures as aregenerally shown in the Figures, including, but not limited to, antibodyfragments, monoclonal antibodies, bispecific antibodies, minibodies,domain antibodies, synthetic antibodies (sometimes referred to herein as“antibody mimetics”), chimeric antibodies, humanized antibodies,antibody fusions (sometimes referred to as “antibody conjugates”), andfragments of each, respectively.

In one embodiment, the antibody is an antibody fragment, as long as itcontains at least one constant domain which can be engineered to produceheterodimers, such as pI engineering. Other antibody fragments that canbe used include fragments that contain one or more of the CH1, CH2, CH3,hinge and CL domains of the invention that have been pI engineered. Forexample, Fc fusions are fusions of the Fc region (CH2 and CH3,optionally with the hinge region) fused to another protein. A number ofFc fusions are known the art and can be improved by the addition of theheterodimerization variants of the invention. In the present case,antibody fusions can be made comprising CH1; CH1, CH2 and CH3; CH2; CH3;CH2 and CH3; CH1 and CH3, any or all of which can be made optionallywith the hinge region, utilizing any combination of heterodimerizationvariants described herein.

scFv Embodiments

In some embodiments of the present invention, one monomer comprises aheavy chain comprises a scFV linked to an Fc domain, and the othermonomer comprises a heavy chain comprising a Fab linked to an Fc domain,e.g. a “typical” heavy chain, and a light chain. By “Fab” or “Fabregion” as used herein is meant the polypeptide that comprises the VH,CH1, VL, and CL immunoglobulin domains. Fab may refer to this region inisolation, or this region in the context of a full length antibody,antibody fragment or Fab fusion protein. By “Fv” or “Fv fragment” or “Fvregion” as used herein is meant a polypeptide that comprises the VL andVH domains of a single antibody.

Several of the heterodimeric antibody embodiments described herein relyon the use of one or more scFv domains, comprising the variable heavyand variable light chains, covalently linked using a linker, forming anantigen binding domain. Some embodiments herein use “standard” linkers,usually linkers of glycine and serine, as is well known in the art.

The present invention further provides charged scFv linkers, tofacilitate the separation in pI between a first and a second monomer.That is, by incorporating a charged scFv linker, either positive ornegative (or both, in the case of scaffolds that use scFvs on differentmonomers), this allows the monomer comprising the charged linker toalter the pI without making further changes in the Fc domains. Thesecharged linkers can be substituted into any scFv containing standardlinkers. Again, as will be appreciated by those in the art, charged scFvlinkers are used on the correct “strand” or monomer, according to thedesired changes in pI. For example, as discussed herein, to make tripleF format heterodimeric antibody, the original pI of the Fv region foreach of the desired antigen binding domains are calculated, and one ischosen to make an scFv, and depending on the pI, either positive ornegative linkers are chosen.

In addition, in the case of anti-CD3 scFv regions, disulfide bonds canbe engineered into the variable heavy and variable light chains to giveadditional stability. Suitable disulfide sequences in the context ofanti-CD3 scFvs are shown in FIG. 8.

Chimeric and Humanized Antibodies

In some embodiments, the antibody can be a mixture from differentspecies, e.g. a chimeric antibody and/or a humanized antibody. Ingeneral, both “chimeric antibodies” and “humanized antibodies” refer toantibodies that combine regions from more than one species. For example,“chimeric antibodies” traditionally comprise variable region(s) from amouse (or rat, in some cases) and the constant region(s) from a human.“Humanized antibodies” generally refer to non-human antibodies that havehad the variable-domain framework regions swapped for sequences found inhuman antibodies. Generally, in a humanized antibody, the entireantibody, except the CDRs, is encoded by a polynucleotide of humanorigin or is identical to such an antibody except within its CDRs. TheCDRs, some or all of which are encoded by nucleic acids originating in anon-human organism, are grafted into the beta-sheet framework of a humanantibody variable region to create an antibody, the specificity of whichis determined by the engrafted CDRs. The creation of such antibodies isdescribed in, e.g., WO 92/11018, Jones, 1986, Nature 321:522-525,Verhoeyen et al., 1988, Science 239:1534-1536, all entirely incorporatedby reference. “Backmutation” of selected acceptor framework residues tothe corresponding donor residues is often required to regain affinitythat is lost in the initial grafted construct (U.S. Pat. No. 5,530,101;U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,761; U.S. Pat. No.5,693,762; U.S. Pat. No. 6,180,370; U.S. Pat. No. 5,859,205; U.S. Pat.No. 5,821,337; U.S. Pat. No. 6,054,297; U.S. Pat. No. 6,407,213, allentirely incorporated by reference). The humanized antibody optimallyalso will comprise at least a portion of an immunoglobulin constantregion, typically that of a human immunoglobulin, and thus willtypically comprise a human Fc region. Humanized antibodies can also begenerated using mice with a genetically engineered immune system. Roqueet al., 2004, Biotechnol. Prog. 20:639-654, entirely incorporated byreference. A variety of techniques and methods for humanizing andreshaping non-human antibodies are well known in the art (See Tsurushita& Vasquez, 2004, Humanization of Monoclonal Antibodies, MolecularBiology of B Cells, 533-545, Elsevier Science (USA), and referencescited therein, all entirely incorporated by reference). Humanizationmethods include but are not limited to methods described in Jones etal., 1986, Nature 321:522-525; Riechmann et al., 1988; Nature332:323-329; Verhoeyen et al., 1988, Science, 239:1534-1536; Queen etal., 1989, Proc Natl Acad Sci, USA 86:10029-33; He et al., 1998, J.Immunol. 160: 1029-1035; Carter et al., 1992, Proc Natl Acad Sci USA89:4285-9, Presta et al., 1997, Cancer Res. 57(20):4593-9; Gorman etal., 1991, Proc. Natl. Acad. Sci. USA 88:4181-4185; O'Connor et al.,1998, Protein Eng 11:321-8, all entirely incorporated by reference.Humanization or other methods of reducing the immunogenicity of nonhumanantibody variable regions may include resurfacing methods, as describedfor example in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA91:969-973, entirely incorporated by reference. In one embodiment, theparent antibody has been affinity matured, as is known in the art.Structure-based methods may be employed for humanization and affinitymaturation, for example as described in U.S. Ser. No. 11/004,590.Selection based methods may be employed to humanize and/or affinitymature antibody variable regions, including but not limited to methodsdescribed in Wu et al., 1999, J. Mol. Biol. 294:151-162; Baca et al.,1997, J. Biol. Chem. 272(16):10678-10684; Rosok et al., 1996, J. Biol.Chem. 271(37): 22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci.USA 95: 8910-8915; Krauss et al., 2003, Protein Engineering16(10):753-759, all entirely incorporated by reference. Otherhumanization methods may involve the grafting of only parts of the CDRs,including but not limited to methods described in U.S. Ser. No.09/810,510; Tan et al., 2002, J. Immunol. 169:1119-1125; De Pascalis etal., 2002, J. Immunol. 169:3076-3084, all entirely incorporated byreference.

Heterodimeric Heavy Chain Constant Regions

Accordingly, the present invention provides heterodimeric proteins basedon the use of monomers containing variant heavy chain constant regions,and specifically the Fc domains, as a first domain. By “monomer” hereinis meant one half of the heterodimeric protein. It should be noted thattraditional antibodies are actually tetrameric (two heavy chains and twolight chains). In the context of the present invention, one pair ofheavy-light chains (if applicable, e.g. if the monomer comprises an Fab)is considered a “monomer”. Similarly, a heavy chain region comprisingthe scFv is considered a monomer. In the case where an Fv region is onefusion partner (e.g. heavy and light variable domains) and anon-antibody protein is another fusion partner, each “half” isconsidered a monomer. Essentially, each monomer comprises sufficientheavy chain constant region to allow heterodimerization engineering,whether that be all the constant region, e.g. Ch1-hinge-CH2-CH3, the Fcregion (CH2-CH3), or just the CH3 domain.

The variant heavy chain constant regions can comprise all or part of theheavy chain constant region, including the full length construct,CH1-hinge-CH2-CH3, or portions thereof, including for example CH2-CH3 orCH3 alone. In addition, the heavy chain region of each monomer can bethe same backbone (CH1-hinge-CH2-CH3 or CH2-CH3) or different. N- andC-terminal truncations and additions are also included within thedefinition; for example, some pI variants include the addition ofcharged amino acids to the C-terminus of the heavy chain domain.

Thus, in general, one monomer of the present “triple F” construct is ascFv region-hinge-Fc domain) and the other is (VH-CH1-hinge-CH2-CH3 plusassociated light chain), with heterodimerization variants, includingsteric, isotypic, charge steering, and pI variants, Fc and FcRnvariants, ablation variants, and additional antigen binding domains(with optional linkers) included in these regions.

In addition to the heterodimerization variants (e.g. steric and pIvariants) outlined herein, the heavy chain regions may also containadditional amino acid substitutions, including changes for altering FcγRand FcRn binding as discussed below.

In addition, some monomers can utilize linkers between the variant heavychain constant region and the fusion partner. For the scFv portion ofthe “bottle-opener”, standard linkers as are known in the art can beused, or the charged scFv linkers described herein. In the case whereadditional fusion partners are made (e.g. FIGS. 1A-1M and 2A-2U),traditional peptide linkers can be used, including flexible linkers ofglycine and serine, or the charged linkers of FIG. 9. In some cases, thelinkers for use as components of the monomer are different from thosedefined below for the ADC constructs, and are in many embodiments notcleavable linkers (such as those susceptible to proteases), althoughcleavable linkers may find use in some embodiments.

The heterodimerization variants include a number of different types ofvariants, including, but not limited to, steric variants (includingcharge variants) and pI variants, that can be optionally andindependently combined with any other variants. In these embodiments, itis important to match “monomer A” with “monomer B”; that is, if aheterodimeric protein relies on both steric variants and pI variants,these need to be correctly matched to each monomer: e.g. the set ofsteric variants that work (1 set on monomer A, 1 set on monomer B) iscombined with pI variant sets (1 set on monomer A, 1 set on monomer B),such that the variants on each monomer are designed to achieve thedesired function, keeping in mind the pI “strandedness” such that stericvariants that may alter pI are put on the appropriate monomer.

It is important to note that the heterodimerization variants outlinedherein (for example, including but not limited to those variants shownin FIGS. 3A-3C and 12A-12J), can be optionally and independentlycombined with any other variants, and on any other monomer. That is,what is important for the heterodimerization is that there are “sets” ofvariants, one set for one monomer and one set for the other. Whetherthese are combined from the FIGS. 1A to 1M (e.g. monomer 1 listings cango together) or switched (monomer 1 pI variants with monomer 2 stericvariants) is irrelevant. However, as noted herein, “strandedness” shouldbe preserved when combinations are made as outlined above. Furthermore,for the additional Fc variants (such as for FcγR binding, FcRn binding,etc.), either monomer, or both monomers, can include any of the listedvariants, independently and optionally. In some cases, both monomershave the additional variants and in some only one monomer has theadditional variants, or they can be combined.

Heterodimerization Variants

The present invention provides heterodimeric proteins, includingheterodimeric antibodies in a variety of formats, which utilizeheterodimeric variants to allow for heterodimeric formation and/orpurification away from homodimers.

Steric Variants

In some embodiments, the formation of heterodimers can be facilitated bythe addition of steric variants. That is, by changing amino acids ineach heavy chain, different heavy chains are more likely to associate toform the heterodimeric structure than to form homodimers with the sameFc amino acid sequences. Suitable steric variants are included in FIGS.3A-3C, and in FIGS. 12A, 12B, 12C, 12D, 12F and 12G.

One mechanism is generally referred to in the art as “knobs and holes”,referring to amino acid engineering that creates steric influences tofavor heterodimeric formation and disfavor homodimeric formation canalso optionally be used; this is sometimes referred to as “knobs andholes”, as described in U.S. Ser. No. 61/596,846, Ridgway et al.,Protein Engineering 9(7):617 (1996); Atwell et al., J. Mol. Biol. 1997270:26; U.S. Pat. No. 8,216,805, all of which are hereby incorporated byreference in their entirety. The Figures identify a number of “monomerA-monomer B” pairs that rely on “knobs and holes”. In addition, asdescribed in Merchant et al., Nature Biotech. 16:677 (1998), these“knobs and hole” mutations can be combined with disulfide bonds to skewformation to heterodimerization.

An additional mechanism that finds use in the generation of heterodimersis sometimes referred to as “electrostatic steering” as described inGunasekaran et al., J. Biol. Chem. 285(25):19637 (2010), herebyincorporated by reference in its entirety. This is sometimes referred toherein as “charge pairs”. In this embodiment, electrostatics are used toskew the formation towards heterodimerization. As those in the art willappreciate, these may also have an effect on pI, and thus onpurification, and thus could in some cases also be considered pIvariants. However, as these were generated to force heterodimerizationand were not used as purification tools, they are classified as “stericvariants”. These include, but are not limited to, D221E/P228E/L368Epaired with D221R/P228R/K409R (e.g. these are “monomer correspondingsets) and C220E/P228E/368E paired with C220R/E224R/P228R/K409R.

Additional monomer A and monomer B variants that can be combined withother variants, optionally and independently in any amount, such as pIvariants outlined herein or other steric variants that are shown in FIG.37 of US 2012/0149876, the figure and legend and SEQ ID NOs of which areincorporated expressly by reference herein.

In some embodiments, the steric variants outlined herein can beoptionally and independently incorporated with any pI variant (or othervariants such as Fc variants, FcRn variants, etc.) into one or bothmonomers, and can be independently and optionally included or excludedfrom the proteins of the invention.

pI (Isoelectric Point) Variants for Heterodimers

In general, as will be appreciated by those in the art, there are twogeneral categories of pI variants: those that increase the pI of theprotein (basic changes) and those that decrease the pI of the protein(acidic changes). As described herein, all combinations of thesevariants can be done: one monomer may be wild type, or a variant thatdoes not display a significantly different pI from wild-type, and theother can be either more basic or more acidic. Alternatively, eachmonomer is changed, one to more basic and one to more acidic.

Preferred combinations of pI variants are shown in FIGS. 3A-3C and 12E.

Heavy Chain pI Changes

A number of pI variants are shown in FIGS. 54A-54C and 55. As outlinedherein and shown in the figures, these changes are shown relative toIgG1, but all isotypes can be altered this way, as well as isotypehybrids. In the case where the heavy chain constant domain is fromIgG2-4, R133E and R133Q can also be used.

Antibody Heterodimers Light Chain Variants

In the case of antibody based heterodimers, e.g. where at least one ofthe monomers comprises a light chain in addition to the heavy chaindomain, pI variants can also be made in the light chain. Amino acidsubstitutions for lowering the pI of the light chain include, but arenot limited to, K126E, K126Q, K145E, K145Q, N152D, S156E, K169E, S202E,K207E and adding peptide DEDE at the c-terminus of the light chain.Changes in this category based on the constant lambda light chaininclude one or more substitutions at R108Q, Q124E, K126Q, N138D, K145Tand Q199E. In addition, increasing the pI of the light chains can alsobe done.

Isotypic Variants

In addition, many embodiments of the invention rely on the “importation”of pI amino acids at particular positions from one IgG isotype intoanother, thus reducing or eliminating the possibility of unwantedimmunogenicity being introduced into the variants. A number of these areshown in FIGS. 10A and 10B. That is, IgG1 is a common isotype fortherapeutic antibodies for a variety of reasons, including high effectorfunction. However, the heavy constant region of IgG1 has a higher pIthan that of IgG2 (8.10 versus 7.31). By introducing IgG2 residues atparticular positions into the IgG1 backbone, the pI of the resultingmonomer is lowered (or increased) and additionally exhibits longer serumhalf-life. For example, IgG1 has a glycine (pI 5.97) at position 137,and IgG2 has a glutamic acid (pI 3.22); importing the glutamic acid willaffect the pI of the resulting protein. As is described below, a numberof amino acid substitutions are generally required to significant affectthe pI of the variant antibody. However, it should be noted as discussedbelow that even changes in IgG2 molecules allow for increased serumhalf-life.

In other embodiments, non-isotypic amino acid changes are made, eitherto reduce the overall charge state of the resulting protein (e.g. bychanging a higher pI amino acid to a lower pI amino acid), or to allowaccommodations in structure for stability, etc. as is more furtherdescribed below.

In addition, by pI engineering both the heavy and light constantdomains, significant changes in each monomer of the heterodimer can beseen. As discussed herein, having the pIs of the two monomers differ byat least 0.5 can allow separation by ion exchange chromatography orisoelectric focusing, or other methods sensitive to isoelectric point.

Calculating pI

The pI of each monomer can depend on the pI of the variant heavy chainconstant domain and the pI of the total monomer, including the variantheavy chain constant domain and the fusion partner. Thus, in someembodiments, the change in pI is calculated on the basis of the variantheavy chain constant domain, using the chart in FIG. 53. As discussedherein, which monomer to engineer is generally decided by the inherentpI of the Fv and scaffold regions. Alternatively, the pI of each monomercan be compared.

Heterodimeric Fc Fusion Proteins

In addition to heterodimeric antibodies, the invention providesheterodimeric proteins that comprise a first monomer comprising avariant Fc region and a first fusion partner and a second monomer, alsocomprising a variant Fc region and a second fusion partner. The variantFc regions are engineered as herein for antibodies, and are thusdifferent, and in general the first and second fusion partners aredifferent as well. In some cases, where one monomer is antibody based(e.g. either comprising a standard heavy and light chain or a Fc domainwith an scFv) and the other is an Fc fusion protein, the resultingheterodimeric protein is called a “fusionbody”.

pI Variants that Also Confer Better FcRn In Vivo Binding

In the case where the pI variant decreases the pI of the monomer, theycan have the added benefit of improving serum retention in vivo.

Although still under examination, Fc regions are believed to have longerhalf-lives in vivo, because binding to FcRn at pH 6 in an endosomesequesters the Fc (Ghetie and Ward, 1997 Immunol Today. 18(12): 592-598,entirely incorporated by reference). The endosomal compartment thenrecycles the Fc to the cell surface. Once the compartment opens to theextracellular space, the higher pH, ˜7.4, induces the release of Fc backinto the blood. In mice, Dall'Acqua et al. showed that Fc mutants withincreased FcRn binding at pH 6 and pH 7.4 actually had reduced serumconcentrations and the same half life as wild-type Fc (Dall'Acqua et al.2002, J. Immunol. 169:5171-5180, entirely incorporated by reference).The increased affinity of Fc for FcRn at pH 7.4 is thought to forbid therelease of the Fc back into the blood. Therefore, the Fc mutations thatwill increase Fc's half-life in vivo will ideally increase FcRn bindingat the lower pH while still allowing release of Fc at higher pH. Theamino acid histidine changes its charge state in the pH range of 6.0 to7.4. Therefore, it is not surprising to find His residues at importantpositions in the Fc/FcRn complex.

Recently it has been suggested that antibodies with variable regionsthat have lower isoelectric points may also have longer serum half-lives(Igawa et al., 2010 PEDS. 23(5): 385-392, entirely incorporated byreference). However, the mechanism of this is still poorly understood.Moreover, variable regions differ from antibody to antibody. Constantregion variants with reduced pI and extended half-life would provide amore modular approach to improving the pharmacokinetic properties ofantibodies, as described herein.

pI variants that find use in this embodiment, as well as their use forpurification optimization, are disclosed in FIG. 20A-20K.

Combination of Heterodimeric Variants

As will be appreciated by those in the art, all of the recitedheterodimerization variants can be optionally and independently combinedin any way, as long as they retain their “strandedness” or “monomerpartition”. In addition, all of these variants can be combined into anyof the heterodimerization formats.

In the case of pI variants, while embodiments finding particular use areshown in the Figures, other combinations can be generated, following thebasic rule of altering the pI difference between two monomers tofacilitate purification.

Nucleic Acids of the Invention

The invention further provides nucleic acid compositions encoding theheterodimeric proteins of the invention. As will be appreciated by thosein the art, the nucleic acid compositions will depend on the format andscaffold of the heterodimeric protein. Thus, for example, when theformat requires three amino acid sequences, such as for the triple Fformat (e.g. a first amino acid monomer comprising an Fc domain and ascFv, a second amino acid monomer comprising a heavy chain and a lightchain), three nucleic acid sequences can be incorporated into one ormore expression vectors for expression. Similarly, some formats (e.g.dual scFv formats such as disclosed in FIG. 1M) only two nucleic acidsare needed; again, they can be put into one or two expression vectors.

Target Antigens

The heterodimeric proteins of the invention may target virtually anyantigens. The “triple F” format is particularly beneficial for targetingtwo (or more) distinct antigens. (As outlined herein, this targeting canbe any combination of monovalent and divalent binding, depending on theformat). Thus the immunoglobulins herein preferably co-engage two targetantigens, although in some cases, three or four antigens can bemonovalently engaged. Each monomer's specificity can be selected fromthe lists below. While the triple F immunoglobulins described herein areparticularly beneficial for targeting distinct antigens, in some casesit may be beneficial to target only one antigen. That is, each monomermay have specificity for the same antigen.

Particular suitable applications of the heterodimeric proteins hereinare co-target pairs for which it is beneficial or critical to engageeach target antigen monovalently. Such antigens may be, for example,immune receptors that are activated upon immune complexation. Cellularactivation of many immune receptors occurs only by cross-linking,achieved typically by antibody/antigen immune complexes, or via effectorcell to target cell engagement. For some immune receptors, for examplethe CD3 signaling receptor on T cells, activation only upon engagementwith co-engaged target is critical, as nonspecific cross-linking in aclinical setting can elicit a cytokine storm and toxicity.Therapeutically, by engaging such antigens monovalently rather thanmultivalently, using the immunoglobulins herein, such activation occursonly in response to cross-linking only in the microenvironment of theprimary target antigen. The ability to target two different antigenswith different valencies is a novel and useful aspect of the presentinvention. Examples of target antigens for which it may betherapeutically beneficial or necessary to co-engage monovalentlyinclude but are not limited to immune activating receptors such as CD3,FcγRs, toll-like receptors (TLRs) such as TLR4 and TLR9, cytokine,chemokine, cytokine receptors, and chemokine receptors. In manyembodiments, one of the antigen binding sites binds to CD3, and in someembodiments it is the scFv-containing monomer.

Virtually any antigen may be targeted by the immunoglobulins herein,including but not limited to proteins, subunits, domains, motifs, and/orepitopes belonging to the following list of target antigens, whichincludes both soluble factors such as cytokines and membrane-boundfactors, including transmembrane receptors: 17-IA, 4-1BB, 4Dc,6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 Adenosine Receptor, A33, ACE,ACE-2, Activin, Activin A, Activin AB, Activin B, Activin C, ActivinRIA, Activin RIA ALK-2, Activin RIB ALK-4, Activin RIIA, Activin RIIB,ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAM8, ADAM9, ADAMTS,ADAMTS4, ADAMTS5, Addressins, aFGF, ALCAM, ALK, ALK-1, ALK-7,alpha-1-antitrypsin, alpha-V/beta-1 antagonist, ANG, Ang, APAF-1, APE,APJ, APP, APRIL, AR, ARC, ART, Artemin, anti-Id, ASPARTIC, Atrialnatriuretic factor, av/b3 integrin, Ax1, b2M, B7-1, B7-2, B7-H,B-lymphocyte Stimulator (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1,BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM,BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 Osteogenin, BMP-4 BMP-2b,BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA(ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMPs, b-NGF,BOK, Bombesin, Bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC,complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8,Calcitonin, cAMP, carcinoembryonic antigen (CEA), carcinoma-associatedantigen, Cathepsin A, Cathepsin B, Cathepsin C/DPPI, Cathepsin D,Cathepsin E, Cathepsin H, Cathepsin L, Cathepsin O, Cathepsin S,Cathepsin V, Cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12,CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21,CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6,CCL7, CCL8, CCL9/10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5,CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8,CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20,CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67proteins), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54,CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123,CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR,cGMP, CINC, Clostridium botulinum toxin, Clostridium perfringens toxin,CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK,CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5,CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14,CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6,cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, Decayaccelerating factor, des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1,Dnase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR(ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, Enkephalinase, eNOS,Eot, eotaxin1, EpCAM, Ephrin B2/EphB4, EPO, ERCC, E-selectin, ET-1,Factor IIa, Factor VII, Factor VIIIc, Factor IX, fibroblast activationprotein (FAP), Fas, FcR1, FEN-1, Ferritin, FGF, FGF-19, FGF-2, FGF3,FGF-8, FGFR, FGFR-3, Fibrin, FL, FLIP, Flt-3, Flt-4, Folliclestimulating hormone, Fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6,FZD7, FZD8, FZD9, FZD10, G250, Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1,GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7(BMP-12, CDMP-3), GDF-8 (Myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF,GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, Glucagon, Glut4, glycoprotein IIb/IIIa (GP IIb/IIIa), GM-CSF, gp130, gp72, GRO, Growthhormone releasing factor, Hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMVgB envelope glycoprotein, HCMV) gH envelope glycoprotein, HCMV UL,Hemopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2/neu(ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gBglycoprotein, HSV gD glycoprotein, HGFA, High molecular weightmelanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp120 V3loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin,human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309,IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF,IGF binding proteins, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R,IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10,IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha,INF-beta, INF-gamma, Inhibin, iNOS, Insulin A-chain, Insulin B-chain,Insulin-like growth factor 1, integrin alpha2, integrin alpha3, integrinalpha4, integrin alpha4/beta1, integrin alpha4/beta7, integrin alpha5(alphaV), integrin alpha5/beta1, integrin alpha5/beta3, integrin alpha6,integrin beta1, integrin beta2, interferon gamma, IP-10, I-TAC, JE,Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein 11, Kallikrein 12,Kallikrein 14, Kallikrein 15, Kallikrein L1, Kallikrein L2, KallikreinL3, Kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), laminin 5,LAMP, LAP, LAP (TGF-1), Latent TGF-1, Latent TGF-1 bp1, LBP, LDGF,LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3,Lfo, LIF, LIGHT, lipoproteins, LIX, LKN, Lptn, L-Selectin, LT-a, LT-b,LTB4, LTBP-1, Lung surfactant, Luteinizing hormone, Lymphotoxin BetaReceptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, MCSF,MDC, Mer, METALLOPROTEASES, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG,MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13,MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo,MSK, MSP, mucin (Muc1), MUC18, Muellerian-inhibitin substance, Mug,MuSK, NAIP, NAP, NCAD, N-Cadherin, NCA 90, NCAM, NCAM, Neprilysin,Neurotrophin-3,-4, or -6, Neurturin, Neuronal growth factor (NGF), NGFR,NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM,OX40L, OX40R, p150, p95, PADPr, Parathyroid hormone, PARC, PARP, PBR,PBSF, PCAD, P-Cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4, PGE,PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), P1GF,PLP, PP14, Proinsulin, Prorelaxin, Protein C, PS, PSA, PSCA, prostatespecific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK,RANKL, RANTES, RANTES, Relaxin A-chain, Relaxin B-chain, renin,respiratory syncytial virus (RSV) F, RSV Fgp, Ret, Rheumatoid factors,RLIP76, RPA2, RSK, S100, SCF/KL, SDF-1, SERINE, Serum albumin, sFRP-3,Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat,STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72),TARC, TCA-3, T-cell receptors (e.g., T-cell receptor alpha/beta), TdT,TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkalinephosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific,TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII,TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, Thrombin, ThymusCk-1, Thyroid stimulating hormone, Tie, TIMP, TIQ, Tissue Factor,TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta2, TNFc,TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1Apo-2, DR4), TNFRSF 10B (TRAILR2DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3DcR1, LIT, TRID),TNFRSF10D (TRAIL R4DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R),TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI),TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF 16(NGFR p75NTR), TNFRSF 17 (BCMA), TNFRSF 18 (GITR AITR), TNFRSF19 (TROYTAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RI CD120a, p55-60),TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNFRIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50),TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3M68, TR6), TNFRSF7(CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6),TNFRSF22 (DcTRAIL R2TNFRH2), TNFRST23 (DcTRAIL R1TNFRH1), TNFRSF25(DR3Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 Ligand, TL2),TNFSF11 (TRANCE/RANK Ligand ODF, OPG Ligand), TNFSF12 (TWEAK Apo-3Ligand, DR3Ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1,THANK, TNFSF20), TNFSF14 (LIGHT HVEM Ligand, LTg), TNFSF15 (TL1A/VEGI),TNFSF18 (GITR Ligand AITR Ligand, TL6), TNFSF1A (TNF-a Conectin, DIF,TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4(OX40 Ligand gp34, TXGP1), TNFSF5 (CD40 Ligand CD154, gp39, HIGM1, IMD3,TRAP), TNFSF6 (Fas Ligand Apo-1 Ligand, APT1 Ligand), TNFSF7 (CD27Ligand CD70), TNFSF8 (CD30 Ligand CD153), TNFSF9 (4-1BB Ligand CD137Ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE,transferring receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associatedantigen CA 125, tumor-associated antigen expressing Lewis Y relatedcarbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, Urokinase, VCAM, VCAM-1,VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3(flt-4), VEGI, VIM, Viral antigens, VLA, VLA-1, VLA-4, VNR integrin, vonWillebrands factor, WIF-1, WNT1, WNT2, WNT2B/13, WNT3, WNT3A, WNT4,WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B,WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD,and receptors for hormones and growth factors. To form the bispecific ortrispecific antibodies of the invention, antibodies to any combinationof these antigens can be made; that is, each of these antigens can beoptionally and independently included or excluded from a multispecificantibody according to the present invention.

Exemplary antigens that may be targeted specifically by theimmunoglobulins of the invention include but are not limited to: CD20,CD19, Her2, EGFR, EpCAM, CD3, FcγRIIIa (CD16), FcγRIIa (CD32a), FcγRIIb(CD32b), FcγRI (CD64), Toll-like receptors (TLRs) such as TLR4 and TLR9,cytokines such as IL-2, IL-5, IL-13, IL-12, IL-23, and TNFα, cytokinereceptors such as IL-2R, chemokines, chemokine receptors, growth factorssuch as VEGF and HGF, and the like. To form the multispecific antibodiesof the invention, antibodies to any combination of these antigens can bemade; that is, each of these antigens can be optionally andindependently included or excluded from a multispecific antibodyaccording to the present invention.

Particularly preferred combinations for bispecific antibodies are anantigen-binding domain to CD3 and an antigen binding domain to CD 19; anantigen-binding domain to CD3 and an antigen binding domain to CD33; anantigen-binding domain to CD3 and an antigen binding domain to CD 38.Again, in many embodiments, the CD3 binding domain is the scFv, havingan exemplary sequence as depicted in the Figures and/or CD3 CDRs asoutlined.

The choice of suitable target antigens and co-targets depends on thedesired therapeutic application. Some targets that have provenespecially amenable to antibody therapy are those with signalingfunctions. Other therapeutic antibodies exert their effects by blockingsignaling of the receptor by inhibiting the binding between a receptorand its cognate ligand. Another mechanism of action of therapeuticantibodies is to cause receptor down regulation. Other antibodies do notwork by signaling through their target antigen. The choice of co-targetswill depend on the detailed biology underlying the pathology of theindication that is being treated.

Monoclonal antibody therapy has emerged as an important therapeuticmodality for cancer (Weiner et al., 2010, Nature Reviews Immunology10:317-327; Reichert et al., 2005, Nature Biotechnology 23[9]:1073-1078;herein expressly incorporated by reference). For anti-cancer treatmentit may be desirable to target one antigen (antigen-1) whose expressionis restricted to the cancerous cells while co-targeting a second antigen(antigen-2) that mediates some immunological killing activity. For othertreatments it may be beneficial to co-target two antigens, for exampletwo angiogenic factors or two growth factors, that are each known toplay some role in proliferation of the tumor. Exemplary co-targets foroncology include but are not limited to HGF and VEGF, IGF-1R and VEGF,Her2 and VEGF, CD19 and CD3, CD20 and CD3, Her2 and CD3, CD19 andFcγRIIIa, CD20 and FcγRIIIa, Her2 and FcγRIIIa. An immunoglobulin of theinvention may be capable of binding VEGF and phosphatidylserine; VEGFand ErbB3; VEGF and PLGF; VEGF and ROBO4; VEGF and BSG2; VEGF and CDCP1;VEGF and ANPEP; VEGF and c-MET; HER-2 and ERB3; HER-2 and BSG2; HER-2and CDCP1; HER-2 and ANPEP; EGFR and CD64; EGFR and BSG2; EGFR andCDCP1; EGFR and ANPEP; IGF1R and PDGFR; IGF1R and VEGF; IGF1R and CD20;CD20 and CD74; CD20 and CD30; CD20 and DR4; CD20 and VEGFR2; CD20 andCD52; CD20 and CD4; HGF and c-MET; HGF and NRP1; HGF andphosphatidylserine; ErbB3 and IGF1R; ErbB3 and IGF1,2; c-Met and Her-2;c-Met and NRP1; c-Met and IGF1R; IGF1,2 and PDGFR; IGF1,2 and CD20;IGF1,2 and IGF1R; IGF2 and EGFR; IGF2 and HER2; IGF2 and CD20; IGF2 andVEGF; IGF2 and IGF1R; IGF1 and IGF2; PDGFRa and VEGFR2; PDGFRa and PLGF;PDGFRa and VEGF; PDGFRa and c-Met; PDGFRa and EGFR; PDGFRb and VEGFR2;PDGFRb and c-Met; PDGFRb and EGFR; RON and c-Met; RON and MTSP1; RON andMSP; RON and CDCP1; VGFR1 and PLGF; VGFR1 and RON; VGFR1 and EGFR;VEGFR2 and PLGF; VEGFR2 and NRP1; VEGFR2 and RON; VEGFR2 and DLL4;VEGFR2 and EGFR; VEGFR2 and ROBO4; VEGFR2 and CD55; LPA and S1P; EPHB2and RON; CTLA4 and VEGF; CD3 and EPCAM; CD40 and IL6; CD40 and IGF; CD40and CD56; CD40 and CD70; CD40 and VEGFR1; CD40 and DR5; CD40 and DR4;CD40 and APRIL; CD40 and BCMA; CD40 and RANKL; CD28 and MAPG; CD80 andCD40; CD80 and CD30; CD80 and CD33; CD80 and CD74; CD80 and CD2; CD80and CD3; CD80 and CD19; CD80 and CD4; CD80 and CD52; CD80 and VEGF; CD80and DR5; CD80 and VEGFR2; CD22 and CD20; CD22 and CD80; CD22 and CD40;CD22 and CD23; CD22 and CD33; CD22 and CD74; CD22 and CD19; CD22 andDR5; CD22 and DR4; CD22 and VEGF; CD22 and CD52; CD30 and CD20; CD30 andCD22; CD30 and CD23; CD30 and CD40; CD30 and VEGF; CD30 and CD74; CD30and CD19; CD30 and DR5; CD30 and DR4; CD30 and VEGFR2; CD30 and CD52;CD30 and CD4; CD138 and RANKL; CD33 and FTL3; CD33 and VEGF; CD33 andVEGFR2; CD33 and CD44; CD33 and DR4; CD33 and DR5; DR4 and CD137; DR4and IGF1,2; DR4 and IGF1R; DR4 and DR5; DR5 and CD40; DR5 and CD137; DR5and CD20; DR5 and EGFR; DR5 and IGF1,2; DR5 and IGFR, DR5 and HER-2, andEGFR and DLL4. Other target combinations include one or more members ofthe EGF/erb-2/erb-3 family.

Other targets (one or more) involved in oncological diseases that theimmunoglobulins herein may bind include, but are not limited to thoseselected from the group consisting of: CD52, CD20, CD19, CD3, CD4, CD8,BMP6, IL12A, IL1A, IL1B, 1L2, IL24, INHA, TNF, TNFSF10, BMP6, EGF, FGF1,FGF10, FGF11, FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2,FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9,GRP, IGF1, IGF2, IL12A, IL1A, IL1B, IL2, INHA, TGFA, TGFB1, TGFB2,TGFB3, VEGF, CDK2, FGF10, FGF18, FGF2, FGF4, FGF7, IGF1R, IL2, BCL2,CD164, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2C, CDKN3, GNRH1,IGFBP6, IL1A, IL1B, ODZ1, PAWR, PLG, TGFB1I1, AR, BRCA1, CDK3, CDK4,CDK5, CDK6, CDK7, CDK9, E2F1, EGFR, ENO1, ERBB2, ESR1, ESR2, IGFBP3,IGFBP6, IL2, INSL4, MYC, NOX5, NR6A1, PAP, PCNA, PRKCQ, PRKD1, PRL,TP53, FGF22, FGF23, FGF9, IGFBP3, IL2, INHA, KLK6, TP53, CHGB, GNRH1,IGF1, IGF2, INHA, INSL3, INSL4, PRL, KLK6, SHBG, NR1D1, NR1H3, NR1I3,NR2F6, NR4A3, ESR1, ESR2, NR0B1, NR0B2, NR1D2, NR1H2, NR1H4, NR112,NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR3C1, NR3C2, NR4A1, NR4A2,NR5A1, NR5A2, NR6 μl, PGR, RARB, FGF1, FGF2, FGF6, KLK3, KRT1, APOC1,BRCA1, CHGA, CHGB, CLU, COL1A1, COL6A1, EGF, ERBB2, ERK8, FGF1, FGF10,FGF11, FGF13, FGF14, FGF16, FGF17, FGF18, FGF2, FGF20, FGF21, FGF22,FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, GNRH1, IGF1, IGF2,IGFBP3, IGFBP6, IL12A, IL1A, IL1B, IL2, IL24, INHA, INSL3, INSL4, KLK10,KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, MMP2, MMP9,MSMB, NTN4, ODZ1, PAP, PLAU, PRL, PSAP, SERPINA3, SHBG, TGFA, TIMP3,CD44, CDH1, CDH10, CDH19, CDH20, CDH7, CDH9, CDH1, CDH10, CDH13, CDH18,CDH19, CDH20, CDH7, CDH8, CDH9, ROBO2, CD44, ILK, ITGA1, APC, CD164,COL6A1, MTSS1, PAP, TGFB1I1, AGR2, AIG1, AKAP1, AKAP2, CANT1, CAV1,CDH12, CLDN3, CLN3, CYB5, CYC1, DAB21P, DES, DNCL1, ELAC2, ENO2, ENO3,FASN, FLJ12584, FLJ25530, GAGEB1, GAGEC1, GGT1, GSTP1, HIP 1, HUMCYT2A,IL29, K6HF, KAI1, KRT2A, MIB1, PART1, PATE, PCA3, PIAS2, PIK3CG, PPID,PR1, PSCA, SLC2A2, SLC33 μl, SLC43 μl, STEAP, STEAP2, TPM1, TPM2, TRPC6,ANGPT1, ANGPT2, ANPEP, ECGF1, EREG, FGF1, FGF2, FIGF, FLT1, JAG1, KDR,LAMA5, NRP1, NRP2, PGF, PLXDC1, STAB 1, VEGF, VEGFC, ANGPTL3, BAI1,COL4A3, IL8, LAMA5, NRP1, NRP2, STAB 1, ANGPTL4, PECAM1, PF4, PROK2,SERPINF1, TNFAIP2, CCL11, CCL2, CXCL1, CXCL10, CXCL3, CXCL5, CXCL6,CXCL9, IFNA1, IFNB1, IFNG, IL1B, IL6, MDK, EDG1, EFNA1, EFNA3, EFNB2,EGF, EPHB4, FGFR3, HGF, IGF1, ITGB3, PDGFA, TEK, TGFA, TGFB1, TGFB2,TGFBR1, CCL2, CDH5, COL1A1, EDG1, ENG, ITGAV, ITGB3, THBS1, THBS2, BAD,BAG1, BCL2, CCNA1, CCNA2, CCND1, CCNE1, CCNE2, CDH1 (E-cadherin), CDKN1B(p27Kip1), CDKN2A (p161NK4a), COL6A1, CTNNB1 (b-catenin), CTSB(cathepsin B), ERBB2 (Her-2), ESR1, ESR2, F3 (TF), FOSL1 (FRA-1), GATA3,GSN (Gelsolin), IGFBP2, IL2RA, IL6, IL6R, IL6ST (glycoprotein 130),ITGA6 (a6 integrin), JUN, KLK5, KRT19, MAP2K7 (c-Jun), MKI67 (Ki-67),NGFB (GF), NGFR, NME1 (M23A), PGR, PLAU (uPA), PTEN, SERPINB5 (maspin),SERPINE1 (PAI-1), TGFA, THBS1 (thrombospondin-1), TIE (Tie-1), TNFRSF6(Fas), TNFSF6 (FasL), TOP2A (topoisomerase Iia), TP53, AZGP1(zinc-a-glycoprotein), BPAG1 (plectin), CDKN1A (p21Wap1/Cip1), CLDN7(claudin-7), CLU (clusterin), ERBB2 (Her-2), FGF1, FLRT1 (fibronectin),GABRP (GABAa), GNAS1, ID2, ITGA6 (a6 integrin), ITGB4 (b 4 integrin),KLF5 (GC Box BP), KRT19 (Keratin 19), KRTHB6 (hair-specific type IIkeratin), MACMARCKS, MT3 (metallothionectin-III), MUC1 (mucin), PTGS2(COX-2), RAC2 (p21Rac2), S100A2, SCGB1D2 (lipophilin B), SCGB2A1(mammaglobin 2), SCGB2A2 (mammaglobin 1), SPRR1B (Spr1), THBS1, THBS2,THBS4, and TNFAIP2 (B94), RON, c-Met, CD64, DLL4, PLGF, CTLA4,phophatidylserine, ROBO4, CD80, CD22, CD40, CD23, CD28, CD80, CD55,CD38, CD70, CD74, CD30, CD138, CD56, CD33, CD2, CD137, DR4, DR5, RANKL,VEGFR2, PDGFR, VEGFR1, MTSP1, MSP, EPHB2, EPHA1, EPHA2, EpCAM, PGE2,NKG2D, LPA, SIP, APRIL, BCMA, MAPG, FLT3, PDGFR alpha, PDGFR beta, ROR1,PSMA, PSCA, SCD1, and CD59. To form the bispecific or trispecificantibodies of the invention, antibodies to any combination of theseantigens can be made; that is, each of these antigens can be optionallyand independently included or excluded from a multispecific antibodyaccording to the present invention.

Monoclonal antibody therapy has become an important therapeutic modalityfor treating autoimmune and inflammatory disorders (Chan & Carter, 2010,Nature Reviews Immunology 10:301-316; Reichert et al., 2005, NatureBiotechnology 23[9]:1073-1078; herein expressly incorporated byreference). Many proteins have been implicated in general autoimmune andinflammatory responses, and thus may be targeted by the immunoglobulinsof the invention. Autoimmune and inflammatory targets include but arenot limited to C5, CCL1 (I-309), CCL11 (eotaxin), CCL13 (mcp-4), CCL15(MIP-1d), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19, CCL2(mcp-1), CCL20 (MIP-3a), CCL21 (MIP-2), CCL23 (MPIF-1), CCL24(MPIF-2/eotaxin-2), CCL25 (TECK), CCL26, CCL3 (MIP-1a), CCL4 (MIP-1b),CCL5 (RANTES), CCL7 (mcp-3), CCL8 (mcp-2), CXCL1, CXCL10 (IP-10), CXCL11(1-TAC/IP-9), CXCL12 (SDF1), CXCL13, CXCL14, CXCL2, CXCL3, CXCL5(ENA-78/LIX), CXCL6 (GCP-2), CXCL9, IL13, IL8, CCL13 (mcp-4), CCR1,CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CR1, IL8RA, XCR1(CCXCR1), IFNA2, IL10, IL13, IL17C, IL1A, IL1B, IL1F10, IL1F5, IL1F6,IL1F7, IL1F8, IL1F9, IL22, IL5, IL8, IL9, LTA, LTB, MIF, SCYE1(endothelial Monocyte-activating cytokine), SPP1, TNF, TNFSF5, IFNA2,IL10RA, IL10RB, IL13, IL13RA1, IL5RA, IL9, IL9R, ABCF1, BCL6, C3, C4A,CEBPB, CRP, ICEBERG, IL1R1, IL1RN, IL8RB, LTB4R, TOLLIP, FADD, IRAK1,IRAK2, MYD88, NCK2, TNFAIP3, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5,TRAF6, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, CD28, CD3E, CD3G, CD3Z,CD69, CD80, CD86, CNR1, CTLA4, CYSLTR1, FCER1A, FCER2, FCGR3A, GPR44,HAVCR2, OPRD1, P2RX7, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9,TLR10, BLR1, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL13,CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24,CCL25, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CL1,CX3CR1, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL10, CXCL11, CXCL12,CXCL13, CXCR4, GPR2, SCYE1, SDF2, XCL1, XCL2, XCR1, AMH, AMHR2, BMPR1A,BMPR1B, BMPR2, C19orf10 (IL27w), CER1, CSF1, CSF2, CSF3, DKFZp451J0118,FGF2, GFI1, IFNA1, IFNB1, IFNG, IGF1, IL1A, IL1B, IL1R1, IL1R2, IL2,IL2RA, IL2RB, IL2RG, IL3, IL4, IL4R, IL5, IL5RA, IL6, IL6R, IL6ST, IL7,IL8, IL8RA, IL8RB, IL9, IL9R, IL10, IL10RA, IL10RB, IL11, IL12RA, IL12A,IL12B, IL12RB1, IL12RB2, IL13, IL13RA1, IL13RA2, IL15, IL15RA, IL16,IL17, IL17R, IL18, IL18R1, IL19, IL20, KITLG, LEP, LTA, LTB, LTB4R,LTB4R2, LTBR, MIF, NPPB, PDGFB, TBX21, TDGF1, TGFA, TGFB1, TGFB1I1,TGFB2, TGFB3, TGFB1, TGFBR1, TGFBR2, TGFBR3, TH1L, TNF, TNFRSF1A,TNFRSF1B, TNFRSF7, TNFRSF8, TNFRSF9, TNFRSF11A, TNFRSF21, TNFSF4,TNFSF5, TNFSF6, TNFSF11, VEGF, ZFPM2, and RNF110 (ZNF144). To form thebispecific or trispecific antibodies of the invention, antibodies to anycombination of these antigens can be made; that is, each of theseantigens can be optionally and independently included or excluded from amultispecific antibody according to the present invention.

Exemplary co-targets for autoimmune and inflammatory disorders includebut are not limited to IL-1 and TNFalpha, IL-6 and TNFalpha, IL-6 andIL-1, IgE and IL-13, IL-1 and IL-13, IL-4 and IL-13, IL-5 and IL-13,IL-9 and IL-13, CD19 and FcγRIIb, and CD79 and FcγRIIb.

Immunoglobulins of the invention with specificity for the followingpairs of targets to treat inflammatory disease are contemplated: TNF andIL-17A; TNF and RANKL; TNF and VEGF; TNF and SOST; TNF and DKK; TNF andalphaVbeta3; TNF and NGF; TNF and IL-23p19; TNF and IL-6; TNF and SOST;TNF and IL-6R; TNF and CD-20; IgE and IL-13; IL-13 and IL23p19; IgE andIL-4; IgE and IL-9; IgE and IL-9; IgE and IL-13; IL-13 and IL-9; IL-13and IL-4; IL-13 and IL-9; IL-13 and IL-9; IL-13 and IL-4; IL-13 andIL-23p19; IL-13 and IL-9; IL-6R and VEGF; IL-6R and IL-17A; IL-6R andRANKL; IL-17A and IL-1beta; IL-1beta and RANKL; IL-1beta and VEGF; RANKLand CD-20; IL-1alpha and IL-1beta; IL-1alpha and IL-1beta.

Pairs of targets that the immunoglobulins described herein can bind andbe useful to treat asthma may be determined. In an embodiment, suchtargets include, but are not limited to, IL-13 and IL-1beta, sinceIL-1beta is also implicated in inflammatory response in asthma; IL-13and cytokines and chemokines that are involved in inflammation, such asIL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-5; IL-13 and IL-25; IL-13and TARC; IL-13 and MDC; IL-13 and MIF; IL-13 and TGF-β; IL-13 and LHRagonist; IL-13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; and IL-13and ADAM8. The immunoglobulins herein may have specificity for one ormore targets involved in asthma selected from the group consisting ofCSF1 (MCSF), CSF2 (GM-CSF), CSF3 (GCSF), FGF2, IFNA1, IFNB1, IFNG,histamine and histamine receptors, IL1A, IL1B, IL2, IL3, IL4, IL5, IL6,IL7, IL8, IL9, IL10, IL11, IL12A, IL12B, IL13, IL14, IL15, IL16, IL17,IL18, IL19, KITLG, PDGFB, IL2RA, IL4R, IL5RA, IL8RA, IL8RB, IL12RB1,IL12RB2, IL13RA1, IL13RA2, IL18R1, TSLP, CCLi, CCL2, CCL3, CCL4, CCL5,CCL7, CCL8, CCL13, CCL17, CCL18, CCL19, CCL20, CCL22, CCL24, CX3CL1,CXCL1, CXCL2, CXCL3, XCLi, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8,CX3CR1, GPR2, XCR1, FOS, GATA3, JAK1, JAK3, STAT6, TBX21, TGFB1, TNF,TNFSF6, YY1, CYSLTR1, FCER1A, FCER2, LTB4R, TB4R2, LTBR, and Chitinase.To form the bispecific or trispecific antibodies of the invention,antibodies to any combination of these antigens can be made; that is,each of these antigens can be optionally and independently included orexcluded from a multispecific antibody according to the presentinvention.

Pairs of targets involved in rheumatoid arthritis (RA) may beco-targeted by the invention, including but not limited to TNF andIL-18; TNF and IL-12; TNF and IL-23; TNF and IL-1beta; TNF and MIF; TNFand IL-17; and TNF and IL-15.

Antigens that may be targeted in order to treat systemic lupuserythematosus (SLE) by the immunoglobulins herein include but are notlimited to CD-20, CD-22, CD-19, CD28, CD4, CD80, HLA-DRA, IL10, IL2,IL4, TNFRSF5, TNFRSF6, TNFSF5, TNFSF6, BLR1, HDAC4, HDAC5, HDAC7A,HDAC9, ICOSL, IGBP1, MS4A1, RGSI, SLA2, CD81, IFNB1, IL10, TNFRSF5,TNFRSF7, TNFSF5, AICDA, BLNK, GALNAC4S-6ST, HDAC4, HDAC5, HDAC7A, HDAC9,IL10, IL11, IL4, INHA, INHBA, KLF6, TNFRSF7, CD28, CD38, CD69, CD80,CD83, CD86, DPP4, FCER2, IL2RA, TNFRSF8, TNFSF7, CD24, CD37, CD40, CD72,CD74, CD79A, CD79B, CR2, ILIR2, ITGA2, ITGA3, MS4A1, ST6GALI, CDIC,CHSTIO, HLA-A, HLA-DRA, and NT5E.; CTLA4, B7.1, B7.2, B1yS, BAFF, C5,IL-4, IL-6, IL-10, IFN-α, and TNF-α. To form the bispecific ortrispecific antibodies of the invention, antibodies to any combinationof these antigens can be made; that is, each of these antigens can beoptionally and independently included or excluded from a multispecificantibody according to the present invention.

The immunoglobulins herein may target antigens for the treatment ofmultiple sclerosis (MS), including but not limited to IL-12, TWEAK,IL-23, CXCL13, CD40, CD40L, IL-18, VEGF, VLA-4, TNF, CD45RB, CD200,IFNgamma, GM-CSF, FGF, C5, CD52, and CCR2. An embodiment includesco-engagement of anti-IL-12 and TWEAK for the treatment of MS.

One aspect of the invention pertains to immunoglobulins capable ofbinding one or more targets involved in sepsis, in an embodiment twotargets, selected from the group consisting TNF, IL-1, MIF, IL-6, IL-8,IL-18, IL-12, IL-23, FasL, LPS, Toll-like receptors, TLR-4, tissuefactor, MIP-2, ADORA2A, CASP1, CASP4, IL-10, IL-1B, NFκB1, PROC,TNFRSFIA, CSF3, CCR3, ILIRN, MIF, NFκB1, PTAFR, TLR2, TLR4, GPR44,HMOX1, midkine, IRAK1, NFκB2, SERPINA1, SERPINE1, and TREM1. To form thebispecific or trispecific antibodies of the invention, antibodies to anycombination of these antigens can be made; that is, each of theseantigens can be optionally and independently included or excluded from amultispecific antibody according to the present invention.

In some cases, immunoglobulins herein may be directed against antigensfor the treatment of infectious diseases.

Antigen Binding Domains

As will be appreciated by those in the art, there are two basic types ofantigen binding domains, those that resemble antibody antigen bindingdomains (e.g. comprising a set of 6 CDRs) and those that can be ligandsor receptors, for example, that bind to targets without the use of CDRs.

Modified Antibodies

In addition to the modifications outlined above, other modifications canbe made. For example, the molecules may be stabilized by theincorporation of disulphide bridges linking the VH and VL domains(Reiter et al., 1996, Nature Biotech. 14:1239-1245, entirelyincorporated by reference). In addition, there are a variety of covalentmodifications of antibodies that can be made as outlined below.

Covalent modifications of antibodies are included within the scope ofthis invention, and are generally, but not always, donepost-translationally. For example, several types of covalentmodifications of the antibody are introduced into the molecule byreacting specific amino acid residues of the antibody with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesmay also be derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole and the like.

In addition, modifications at cysteines are particularly useful inantibody-drug conjugate (ADC) applications, further described below. Insome embodiments, the constant region of the antibodies can beengineered to contain one or more cysteines that are particularly “thiolreactive”, so as to allow more specific and controlled placement of thedrug moiety. See for example U.S. Pat. No. 7,521,541, incorporated byreference in its entirety herein.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing alpha-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pKa of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using 125I or 131I to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method described abovebeing suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R′—N═C═N—R′), where R and R′ are optionallydifferent alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinkingantibodies to a water-insoluble support matrix or surface for use in avariety of methods, in addition to methods described below. Commonlyused crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis (succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such ascynomolgusogen bromide-activated carbohydrates and the reactivesubstrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128;4,247,642; 4,229,537; and 4,330,440, all entirely incorporated byreference, are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively.Alternatively, these residues are deamidated under mildly acidicconditions. Either form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 [1983],entirely incorporated by reference), acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

In addition, as will be appreciated by those in the art, labels(including fluorescent, enzymatic, magnetic, radioactive, etc. can allbe added to the antibodies (as well as the other compositions of theinvention).

Glycosylation

Another type of covalent modification is alterations in glycosylation.In another embodiment, the antibodies disclosed herein can be modifiedto include one or more engineered glycoforms. By “engineered glycoform”as used herein is meant a carbohydrate composition that is covalentlyattached to the antibody, wherein said carbohydrate composition differschemically from that of a parent antibody. Engineered glycoforms may beuseful for a variety of purposes, including but not limited to enhancingor reducing effector function. A preferred form of engineered glycoformis afucosylation, which has been shown to be correlated to an increasein ADCC function, presumably through tighter binding to the FcγRIIIareceptor. In this context, “afucosylation” means that the majority ofthe antibody produced in the host cells is substantially devoid offucose, e.g. 90-95-98% of the generated antibodies do not haveappreciable fucose as a component of the carbohydrate moiety of theantibody (generally attached at N297 in the Fc region). Definedfunctionally, afucosylated antibodies generally exhibit at least a 50%or higher affinity to the FcγRIIIa receptor.

Engineered glycoforms may be generated by a variety of methods known inthe art (Umañ a et al., 1999, Nat Biotechnol 17:176-180; Davies et al.,2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473; U.S.Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No. 10/113,929;PCT WO 00/61739A1; PCT WO 01/29246A1; PCT WO 02/31140A1; PCT WO02/30954A1, all entirely incorporated by reference; (Potelligent®technology [Biowa, Inc., Princeton, N.J.]; GlycoMAb® glycosylationengineering technology [Glycart Biotechnology AG, Zürich, Switzerland]).Many of these techniques are based on controlling the level offucosylated and/or bisecting oligosaccharides that are covalentlyattached to the Fc region, for example by expressing an IgG in variousorganisms or cell lines, engineered or otherwise (for example Lec-13 CHOcells or rat hybridoma YB2/0 cells, by regulating enzymes involved inthe glycosylation pathway (for example FUT8 [α1,6-fucosyltranserase]and/or β1-4-N-acetylglucosaminyltransferase III [GnTIII]), or bymodifying carbohydrate(s) after the IgG has been expressed. For example,the “sugar engineered antibody” or “SEA technology” of Seattle Geneticsfunctions by adding modified saccharides that inhibit fucosylationduring production; see for example 20090317869, hereby incorporated byreference in its entirety. Engineered glycoform typically refers to thedifferent carbohydrate or oligosaccharide; thus an antibody can includean engineered glycoform.

Alternatively, engineered glycoform may refer to the IgG variant thatcomprises the different carbohydrate or oligosaccharide. As is known inthe art, glycosylation patterns can depend on both the sequence of theprotein (e.g., the presence or absence of particular glycosylation aminoacid residues, discussed below), or the host cell or organism in whichthe protein is produced. Particular expression systems are discussedbelow.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tri-peptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tri-peptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid,most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tri-peptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thestarting sequence (for O-linked glycosylation sites). For ease, theantibody amino acid sequence is preferably altered through changes atthe DNA level, particularly by mutating the DNA encoding the targetpolypeptide at preselected bases such that codons are generated thatwill translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theantibody is by chemical or enzymatic coupling of glycosides to theprotein. These procedures are advantageous in that they do not requireproduction of the protein in a host cell that has glycosylationcapabilities for N- and O-linked glycosylation. Depending on thecoupling mode used, the sugar(s) may be attached to (a) arginine andhistidine, (b) free carboxyl groups, (c) free sulfhydryl groups such asthose of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in WO 87/05330 and in Aplin andWriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306, both entirelyincorporated by reference.

Removal of carbohydrate moieties present on the starting antibody (e.g.post-translationally) may be accomplished chemically or enzymatically.Chemical deglycosylation requires exposure of the protein to thecompound trifluoromethanesulfonic acid, or an equivalent compound. Thistreatment results in the cleavage of most or all sugars except thelinking sugar (N-acetylglucosamine or N-acetylgalactosamine), whileleaving the polypeptide intact. Chemical deglycosylation is described byHakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 and by Edge etal., 1981, Anal. Biochem. 118:131, both entirely incorporated byreference. Enzymatic cleavage of carbohydrate moieties on polypeptidescan be achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., 1987, Meth. Enzymol. 138:350, entirelyincorporated by reference. Glycosylation at potential glycosylationsites may be prevented by the use of the compound tunicamycin asdescribed by Duskin et al., 1982, J. Biol. Chem. 257:3105, entirelyincorporated by reference. Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

Another type of covalent modification of the antibody comprises linkingthe antibody to various nonproteinaceous polymers, including, but notlimited to, various polyols such as polyethylene glycol, polypropyleneglycol or polyoxyalkylenes, in the manner set forth in, for example,2005-2006 PEG Catalog from Nektar Therapeutics (available at the Nektarwebsite) U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417;4,791,192 or 4,179,337, all entirely incorporated by reference. Inaddition, as is known in the art, amino acid substitutions may be madein various positions within the antibody to facilitate the addition ofpolymers such as PEG. See for example, U.S. Publication No.2005/0114037A1, entirely incorporated by reference.

Additional Fc Variants for Additional Functionality

In addition to pI amino acid variants, there are a number of useful Fcamino acid modification that can be made for a variety of reasons,including, but not limited to, altering binding to one or more FcγRreceptors, altered binding to FcRn receptors, etc.

Accordingly, the proteins of the invention can include amino acidmodifications, including the heterodimerization variants outlinedherein, which includes the pI variants and steric variants. Each set ofvariants can be independently and optionally included or excluded fromany particular heterodimeric protein.

FcγR Variants

Accordingly, there are a number of useful Fc substitutions that can bemade to alter binding to one or more of the FcγR receptors.Substitutions that result in increased binding as well as decreasedbinding can be useful. For example, it is known that increased bindingto Fc RIIIa generally results in increased ADCC (antibody dependentcell-mediated cytotoxicity; the cell-mediated reaction whereinnonspecific cytotoxic cells that express FcγRs recognize bound antibodyon a target cell and subsequently cause lysis of the target cell).Similarly, decreased binding to FcγRIIb (an inhibitory receptor) can bebeneficial as well in some circumstances Amino acid substitutions thatfind use in the present invention include those listed in U.S. Ser. No.11/124,620 (particularly FIG. 41), Ser. Nos. 11/174,287, 11/396,495,11/538,406, all of which are expressly incorporated herein by referencein their entirety and specifically for the variants disclosed therein.Particular variants that find use include, but are not limited to, 236A,239D, 239E, 332E, 332D, 239D/332E, 267D, 267E, 328F, 267E/328F,236A/332E, 239D/332E/330Y, 239D, 332E/330L, 243A, 243L, 264A, 264V and299T.

In addition, there are additional Fc substitutions that find use inincreased binding to the FcRn receptor and increased serum half life, asspecifically disclosed in U.S. Ser. No. 12/341,769, hereby incorporatedby reference in its entirety, including, but not limited to, 434S, 434A,428L, 308F, 259I, 428L/434S, 259I/308F, 436I/428L, 436I or V/434S,436V/428L and 259I/308F/428L.

Linkers

The present invention optionally provides linkers as needed, for examplein the addition of additional antigen binding sites, as depicted forexample in FIG. 2A-2U, where “the other end” of the molecule containsadditional antigen binding components. In addition, as outlined below,linkers are optionally also used in antibody drug conjugate (ADC)systems. When used to join the components of the central mAb-Fvconstructs, the linker is generally a polypeptide comprising two or moreamino acid residues joined by peptide bonds and are used to link one ormore of the components of the present invention. Such linkerpolypeptides are well known in the art (see e.g., Holliger, P., et al.(1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al.(1994) Structure 2:1121-1123). A variety of linkers may find use in someembodiments described herein. As will be appreciated by those in theart, there are at least three different linker types used in the presentinvention.

“Linker” herein is also referred to as “linker sequence”, “spacer”,“tethering sequence” or grammatical equivalents thereof. Homo- orhetero-bifunctional linkers as are well known (see, 1994 Pierce ChemicalCompany catalog, technical section on cross-linkers, pages 155-200,incorporated entirely by reference). A number of strategies may be usedto covalently link molecules together. These include, but are notlimited to polypeptide linkages between N- and C-termini of proteins orprotein domains, linkage via disulfide bonds, and linkage via chemicalcross-linking reagents. In one aspect of this embodiment, the linker isa peptide bond, generated by recombinant techniques or peptidesynthesis. The linker peptide may predominantly include the followingamino acid residues: Gly, Ser, Ala, or Thr. The linker peptide shouldhave a length that is adequate to link two molecules in such a way thatthey assume the correct conformation relative to one another so thatthey retain the desired activity. In one embodiment, the linker is fromabout 1 to 50 amino acids in length, preferably about 1 to 30 aminoacids in length. In one embodiment, linkers of 1 to 20 amino acids inlength may be used. Useful linkers include glycine-serine polymers,including for example (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n isan integer of at least one, glycine-alanine polymers, alanine-serinepolymers, and other flexible linkers. Alternatively, a variety ofnonproteinaceous polymers, including but not limited to polyethyleneglycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers ofpolyethylene glycol and polypropylene glycol, may find use as linkers,that is may find use as linkers.

Other linker sequences may include any sequence of any length of CL/CH1domain but not all residues of CL/CH1 domain; for example the first 5-12amino acid residues of the CL/CH1 domains. Linkers can be derived fromimmunoglobulin light chain, for example Cκ or Cλ. Linkers can be derivedfrom immunoglobulin heavy chains of any isotype, including for exampleCγ1, Cγ2, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ. Linker sequences may alsobe derived from other proteins such as Ig-like proteins (e.g. TCR, FcR,KIR), hinge region-derived sequences, and other natural sequences fromother proteins.

Antibody-Drug Conjugates

In some embodiments, the multispecific antibodies of the invention areconjugated with drugs to form antibody-drug conjugates (ADCs). Ingeneral, ADCs are used in oncology applications, where the use ofantibody-drug conjugates for the local delivery of cytotoxic orcytostatic agents allows for the targeted delivery of the drug moiety totumors, which can allow higher efficacy, lower toxicity, etc. Anoverview of this technology is provided in Ducry et al., BioconjugateChem., 21:5-13 (2010), Carter et al., Cancer J. 14(3):154 (2008) andSenter, Current Opin. Chem. Biol. 13:235-244 (2009), all of which arehereby incorporated by reference in their entirety.

Thus the invention provides multispecific antibodies conjugated todrugs. Generally, conjugation is done by covalent attachment to theantibody, as further described below, and generally relies on a linker,often a peptide linkage (which, as described below, may be designed tobe sensitive to cleavage by proteases at the target site or not). Inaddition, as described above, linkage of the linker-drug unit (LU-D) canbe done by attachment to cysteines within the antibody. As will beappreciated by those in the art, the number of drug moieties perantibody can change, depending on the conditions of the reaction, andcan vary from 1:1 to 10:1 drug:antibody. As will be appreciated by thosein the art, the actual number is an average.

Thus the invention provides multispecific antibodies conjugated todrugs. As described below, the drug of the ADC can be any number ofagents, including but not limited to cytotoxic agents such aschemotherapeutic agents, growth inhibitory agents, toxins (for example,an enzymatically active toxin of bacterial, fungal, plant, or animalorigin, or fragments thereof), or a radioactive isotope (that is, aradioconjugate) are provided. In other embodiments, the inventionfurther provides methods of using the ADCs.

Drugs for use in the present invention include cytotoxic drugs,particularly those which are used for cancer therapy. Such drugsinclude, in general, DNA damaging agents, anti-metabolites, naturalproducts and their analogs. Exemplary classes of cytotoxic agentsinclude the enzyme inhibitors such as dihydrofolate reductaseinhibitors, and thymidylate synthase inhibitors, DNA intercalators, DNAcleavers, topoisomerase inhibitors, the anthracycline family of drugs,the vinca drugs, the mitomycins, the bleomycins, the cytotoxicnucleosides, the pteridine family of drugs, diynenes, thepodophyllotoxins, dolastatins, maytansinoids, differentiation inducers,and taxols.

Members of these classes include, for example, methotrexate,methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine,cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin,daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminomycin,aminopterin, tallysomycin, podophyllotoxin and podophyllotoxinderivatives such as etoposide or etoposide phosphate, vinblastine,vincristine, vindesine, taxanes including taxol, taxotere retinoic acid,butyric acid, N8-acetyl spermidine, camptothecin, calicheamicin,esperamicin, ene-diynes, duocarmycin A, duocarmycin SA, calicheamicin,camptothecin, maytansinoids (including DM1), monomethylauristatin E(MMAE), monomethylauristatin F (MMAF), and maytansinoids (DM4) and theiranalogues.

Toxins may be used as antibody-toxin conjugates and include bacterialtoxins such as diphtheria toxin, plant toxins such as ricin, smallmolecule toxins such as geldanamycin (Mandler et al (2000) J. Nat.Cancer Inst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med.Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem.13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl.Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998)Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342).Toxins may exert their cytotoxic and cytostatic effects by mechanismsincluding tubulin binding, DNA binding, or topoisomerase inhibition.

Conjugates of a multispecific antibody and one or more small moleculetoxins, such as a maytansinoids, dolastatins, auristatins, atrichothecene, calicheamicin, and CC1065, and the derivatives of thesetoxins that have toxin activity, are contemplated.

Maytansinoids

Maytansine compounds suitable for use as maytansinoid drug moieties arewell known in the art, and can be isolated from natural sourcesaccording to known methods, produced using genetic engineeringtechniques (see Yu et al (2002) PNAS 99:7968-7973), or maytansinol andmaytansinol analogues prepared synthetically according to known methods.As described below, drugs may be modified by the incorporation of afunctionally active group such as a thiol or amine group for conjugationto the antibody.

Exemplary maytansinoid drug moieties include those having a modifiedaromatic ring, such as: C-19-dechloro (U.S. Pat. No. 4,256,746)(prepared by lithium aluminum hydride reduction of ansamytocin P2);C-20-hydroxy (or C-20-demethyl)+/−C-19-dechloro (U.S. Pat. Nos.4,361,650 and 4,307,016) (prepared by demethylation using Streptomycesor Actinomyces or dechlorination using LAH); and C-20-demethoxy,C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat. No. 4,294,757) (prepared byacylation using acyl chlorides) and those having modifications at otherpositions

Exemplary maytansinoid drug moieties also include those havingmodifications such as: C-9-SH (U.S. Pat. No. 4,424,219) (prepared by thereaction of maytansinol with H2S or P2S5);C-14-alkoxymethyl(demethoxy/CH2OR) (U.S. Pat. No. 4,331,598);C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (U.S. Pat. No.4,450,254) (prepared from Nocardia); C-15-hydroxy/acyloxy (U.S. Pat. No.4,364,866) (prepared by the conversion of maytansinol by Streptomyces);C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated fromTrewia nudlflora); C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and4,322,348) (prepared by the demethylation of maytansinol byStreptomyces); and 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by thetitanium trichloride/LAH reduction of maytansinol).

Of particular use are DM1 (disclosed in U.S. Pat. No. 5,208,020,incorporated by reference) and DM4 (disclosed in U.S. Pat. No.7,276,497, incorporated by reference). See also a number of additionalmaytansinoid derivatives and methods in U.S. Pat. No. 5,416,064,WO/01/24763, U.S. Pat. Nos. 7,303,749, 7,601,354, U.S. Ser. No.12/631,508, WO02/098883, U.S. Pat. Nos. 6,441,163, 7,368,565, WO02/16368and WO04/1033272, all of which are expressly incorporated by referencein their entirety.

ADCs containing maytansinoids, methods of making same, and theirtherapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020;5,416,064; 6,441,163 and European Patent EP 0 425 235 B1, thedisclosures of which are hereby expressly incorporated by reference. Liuet al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) described ADCscomprising a maytansinoid designated DM1 linked to the monoclonalantibody C242 directed against human colorectal cancer. The conjugatewas found to be highly cytotoxic towards cultured colon cancer cells,and showed antitumor activity in an in vivo tumor growth assay.

Chari et al., Cancer Research 52:127-131 (1992) describe ADCs in which amaytansinoid was conjugated via a disulfide linker to the murineantibody A7 binding to an antigen on human colon cancer cell lines, orto another murine monoclonal antibody TA.1 that binds the HER-2/neuoncogene. The cytotoxicity of the TA.1-maytansonoid conjugate was testedin vitro on the human breast cancer cell line SK-BR-3, which expresses3×105 HER-2 surface antigens per cell. The drug conjugate achieved adegree of cytotoxicity similar to the free maytansinoid drug, whichcould be increased by increasing the number of maytansinoid moleculesper antibody molecule. The A7-maytansinoid conjugate showed low systemiccytotoxicity in mice.

Auristatins and Dolastatins

In some embodiments, the ADC comprises a multispecific antibodyconjugated to dolastatins or dolastatin peptidic analogs andderivatives, the auristatins (U.S. Pat. Nos. 5,635,483; 5,780,588).Dolastatins and auristatins have been shown to interfere withmicrotubule dynamics, GTP hydrolysis, and nuclear and cellular division(Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584)and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity(Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965). Thedolastatin or auristatin drug moiety may be attached to the antibodythrough the N (amino) terminus or the C (carboxyl) terminus of thepeptidic drug moiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in “Senter etal, Proceedings of the American Association for Cancer Research, Volume45, Abstract Number 623, presented Mar. 28, 2004 and described in UnitedStates Patent Publication No. 2005/0238648, the disclosure of which isexpressly incorporated by reference in its entirety.

An exemplary auristatin embodiment is MMAE (see U.S. Pat. No. 6,884,869expressly incorporated by reference in its entirety).

Another exemplary auristatin embodiment is MMAF (see US 2005/0238649,U.S. Pat. Nos. 5,767,237 and 6,124,431, expressly incorporated byreference in their entirety).

Additional exemplary embodiments comprising MMAE or MMAF and variouslinker components (described further herein) have the followingstructures and abbreviations (wherein Ab means antibody and p is 1 toabout 8):

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see E. Schroder and K. Lubke, “The Peptides”,volume 1, pp 76-136, 1965, Academic Press) that is well known in thefield of peptide chemistry. The auristatin/dolastatin drug moieties maybe prepared according to the methods of: U.S. Pat. No. 5,635,483; U.S.Pat. No. 5,780,588; Pettit et al (1989) J. Am. Chem. Soc. 111:5463-5465;Pettit et al (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R.,et al. Synthesis, 1996, 719-725; Pettit et al (1996) J. Chem. Soc.Perkin Trans. 1 5:859-863; and Doronina (2003) Nat Biotechnol21(7):778-784.

Calicheamicin

In other embodiments, the ADC comprises an antibody of the inventionconjugated to one or more calicheamicin molecules. For example, Mylotargis the first commercial ADC drug and utilizes calicheamicin γ1 as thepayload (see U.S. Pat. No. 4,970,198, incorporated by reference in itsentirety). Additional calicheamicin derivatives are described in U.S.Pat. Nos. 5,264,586, 5,384,412, 5,550,246, 5,739,116, 5,773,001,5,767,285 and 5,877,296, all expressly incorporated by reference. Thecalicheamicin family of antibiotics are capable of producingdouble-stranded DNA breaks at sub-picomolar concentrations. For thepreparation of conjugates of the calicheamicin family, see U.S. Pat.Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710,5,773,001, 5,877,296 (all to American Cyanamid Company). Structuralanalogues of calicheamicin which may be used include, but are notlimited to, γ1I, α2I, α2I, N-acetyl-γ1I, PSAG and θI1 (Hinman et al.,Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research58:2925-2928 (1998) and the aforementioned U.S. patents to AmericanCyanamid). Another anti-tumor drug that the antibody can be conjugatedis QFA which is an antifolate. Both calicheamicin and QFA haveintracellular sites of action and do not readily cross the plasmamembrane. Therefore, cellular uptake of these agents through antibodymediated internalization greatly enhances their cytotoxic effects.

Duocarmycins

CC-1065 (see U.S. Pat. No. 4,169,888, incorporated by reference) andduocarmycins are members of a family of antitumor antibiotics utilizedin ADCs. These antibiotics appear to work through sequence-selectivelyalkylating DNA at the N3 of adenine in the minor groove, which initiatesa cascade of events that result in apoptosis.

Important members of the duocarmycins include duocarmycin A (U.S. Pat.No. 4,923,990, incorporated by reference) and duocarmycin SA (U.S. Pat.No. 5,101,038, incorporated by reference), and a large number ofanalogues as described in U.S. Pat. Nos. 7,517,903, 7,691,962,5,101,038; 5,641,780; 5,187,186; 5,070,092; 5,070,092; 5,641,780;5,101,038; 5,084,468, 5,475,092, 5,585,499, 5,846,545, WO2007/089149,WO2009/017394A1, U.S. Pat. Nos. 5,703,080, 6,989,452, 7,087,600,7,129,261, 7,498,302, and 7,507,420, all of which are expresslyincorporated by reference.

Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the antibodies of theinvention include BCNU, streptozoicin, vincristine and 5-fluorouracil,the family of agents known collectively LL-E33288 complex described inU.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat.No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an ADC formed between anantibody and a compound with nucleolytic activity (e.g., a ribonucleaseor a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated antibodies. Examples includeAt211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 andradioactive isotopes of Lu.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as Tc99m or I123, Re186, Re188 and In111 can be attached viaa cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun 80: 49-57 can be used to incorporate Iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

For compositions comprising a plurality of antibodies, the drug loadingis represented by p, the average number of drug molecules per Antibody.Drug loading may range from 1 to 20 drugs (D) per Antibody. The averagenumber of drugs per antibody in preparation of conjugation reactions maybe characterized by conventional means such as mass spectroscopy, ELISAassay, and HPLC. The quantitative distribution ofAntibody-Drug-Conjugates in terms of p may also be determined.

In some instances, separation, purification, and characterization ofhomogeneous Antibody-Drug-conjugates where p is a certain value fromAntibody-Drug-Conjugates with other drug loadings may be achieved bymeans such as reverse phase HPLC or electrophoresis. In exemplaryembodiments, p is 2, 3, 4, 5, 6, 7, or 8 or a fraction thereof.

The generation of Antibody-drug conjugate compounds can be accomplishedby any technique known to the skilled artisan. Briefly, theAntibody-drug conjugate compounds can include a multispecific antibodyas the Antibody unit, a drug, and optionally a linker that joins thedrug and the binding agent.

A number of different reactions are available for covalent attachment ofdrugs and/or linkers to binding agents. This is can be accomplished byreaction of the amino acid residues of the binding agent, for example,antibody molecule, including the amine groups of lysine, the freecarboxylic acid groups of glutamic and aspartic acid, the sulfhydrylgroups of cysteine and the various moieties of the aromatic amino acids.A commonly used non-specific methods of covalent attachment is thecarbodiimide reaction to link a carboxy (or amino) group of a compoundto amino (or carboxy) groups of the antibody. Additionally, bifunctionalagents such as dialdehydes or imidoesters have been used to link theamino group of a compound to amino groups of an antibody molecule.

Also available for attachment of drugs to binding agents is the Schiffbase reaction. This method involves the periodate oxidation of a drugthat contains glycol or hydroxy groups, thus forming an aldehyde whichis then reacted with the binding agent. Attachment occurs via formationof a Schiff base with amino groups of the binding agent. Isothiocyanatescan also be used as coupling agents for covalently attaching drugs tobinding agents. Other techniques are known to the skilled artisan andwithin the scope of the present invention.

In some embodiments, an intermediate, which is the precursor of thelinker, is reacted with the drug under appropriate conditions. In otherembodiments, reactive groups are used on the drug and/or theintermediate. The product of the reaction between the drug and theintermediate, or the derivatized drug, is subsequently reacted with anmultispecific antibody of the invention under appropriate conditions.

It will be understood that chemical modifications may also be made tothe desired compound in order to make reactions of that compound moreconvenient for purposes of preparing conjugates of the invention. Forexample a functional group e g amine, hydroxyl, or sulfhydryl, may beappended to the drug at a position which has minimal or an acceptableeffect on the activity or other properties of the drug

ADC Linker Units

Typically, the antibody-drug conjugate compounds comprise a Linker unitbetween the drug unit and the antibody unit. In some embodiments, thelinker is cleavable under intracellular or extracellular conditions,such that cleavage of the linker releases the drug unit from theantibody in the appropriate environment. For example, solid tumors thatsecrete certain proteases may serve as the target of the cleavablelinker; in other embodiments, it is the intracellular proteases that areutilized. In yet other embodiments, the linker unit is not cleavable andthe drug is released, for example, by antibody degradation in lysosomes.

In some embodiments, the linker is cleavable by a cleaving agent that ispresent in the intracellular environment (for example, within a lysosomeor endosome or caveolea). The linker can be, for example, a peptidyllinker that is cleaved by an intracellular peptidase or protease enzyme,including, but not limited to, a lysosomal or endosomal protease. Insome embodiments, the peptidyl linker is at least two amino acids longor at least three amino acids long or more.

Cleaving agents can include, without limitation, cathepsins B and D andplasmin, all of which are known to hydrolyze dipeptide drug derivativesresulting in the release of active drug inside target cells (see, e.g.,Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). Peptidyllinkers that are cleavable by enzymes that are present inCD38-expressing cells. For example, a peptidyl linker that is cleavableby the thiol-dependent protease cathepsin-B, which is highly expressedin cancerous tissue, can be used (e.g., a Phe-Leu or a Gly-Phe-Leu-Glylinker (SEQ ID NO: 460)). Other examples of such linkers are described,e.g., in U.S. Pat. No. 6,214,345, incorporated herein by reference inits entirety and for all purposes.

In some embodiments, the peptidyl linker cleavable by an intracellularprotease is a Val-Cit linker or a Phe-Lys linker (see, e.g., U.S. Pat.No. 6,214,345, which describes the synthesis of doxorubicin with theval-cit linker).

In other embodiments, the cleavable linker is pH-sensitive, that is,sensitive to hydrolysis at certain pH values. Typically, thepH-sensitive linker hydrolyzable under acidic conditions. For example,an acid-labile linker that is hydrolyzable in the lysosome (for example,a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide,orthoester, acetal, ketal, or the like) may be used. (See, e.g., U.S.Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999,Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem.264:14653-14661.) Such linkers are relatively stable under neutral pHconditions, such as those in the blood, but are unstable at below pH 5.5or 5.0, the approximate pH of the lysosome. In certain embodiments, thehydrolyzable linker is a thioether linker (such as, e.g., a thioetherattached to the therapeutic agent via an acylhydrazone bond (see, e.g.,U.S. Pat. No. 5,622,929).

In yet other embodiments, the linker is cleavable under reducingconditions (for example, a disulfide linker). A variety of disulfidelinkers are known in the art, including, for example, those that can beformed using SATA (N-succinimidyl-5-acetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-, SPDB and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res.47:5924-5931; Wawrzynczak et al., In Immunoconjugates: AntibodyConjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed.,Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935.)

In other embodiments, the linker is a malonate linker (Johnson et al.,1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau etal., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3′-N-amide analog(Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

In yet other embodiments, the linker unit is not cleavable and the drugis released by antibody degradation. (See U.S. Publication No.2005/0238649 incorporated by reference herein in its entirety and forall purposes).

In many embodiments, the linker is self-immolative. As used herein, theterm “self-immolative Spacer” refers to a bifunctional chemical moietythat is capable of covalently linking together two spaced chemicalmoieties into a stable tripartite molecule. It will spontaneouslyseparate from the second chemical moiety if its bond to the first moietyis cleaved. See for example, WO 2007059404A2, WO06110476A2,W005112919A2, WO2010/062171, WO09/017,394, WO07/089,149, WO 07/018,431,WO04/043493 and WO02/083180, which are directed to drug-cleavablesubstrate conjugates where the drug and cleavable substrate areoptionally linked through a self-immolative linker and which are allexpressly incorporated by reference.

Often the linker is not substantially sensitive to the extracellularenvironment. As used herein, “not substantially sensitive to theextracellular environment,” in the context of a linker, means that nomore than about 20%, 15%, 10%, 5%, 3%, or no more than about 1% of thelinkers, in a sample of antibody-drug conjugate compound, are cleavedwhen the antibody-drug conjugate compound presents in an extracellularenvironment (for example, in plasma).

Whether a linker is not substantially sensitive to the extracellularenvironment can be determined, for example, by incubating with plasmathe antibody-drug conjugate compound for a predetermined time period(for example, 2, 4, 8, 16, or 24 hours) and then quantitating the amountof free drug present in the plasma.

In other, non-mutually exclusive embodiments, the linker promotescellular internalization. In certain embodiments, the linker promotescellular internalization when conjugated to the therapeutic agent (thatis, in the milieu of the linker-therapeutic agent moiety of theantibody-drug conjugate compound as described herein). In yet otherembodiments, the linker promotes cellular internalization whenconjugated to both the auristatin compound and the multispecificantibodies of the invention.

A variety of exemplary linkers that can be used with the presentcompositions and methods are described in WO 2004-010957, U.S.Publication No. 2006/0074008, U.S. Publication No. 20050238649, and U.S.Publication No. 2006/0024317 (each of which is incorporated by referenceherein in its entirety and for all purposes).

Drug Loading

Drug loading is represented by p and is the average number of Drugmoieties per antibody in a molecule. Drug loading (“p”) may be 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or moremoieties (D) per antibody, although frequently the average number is afraction or a decimal. Generally, drug loading of from 1 to 4 isfrequently useful, and from 1 to 2 is also useful. ADCs of the inventioninclude collections of antibodies conjugated with a range of drugmoieties, from 1 to 20. The average number of drug moieties per antibodyin preparations of ADC from conjugation reactions may be characterizedby conventional means such as mass spectroscopy and, ELISA assay.

The quantitative distribution of ADC in terms of p may also bedetermined. In some instances, separation, purification, andcharacterization of homogeneous ADC where p is a certain value from ADCwith other drug loadings may be achieved by means such aselectrophoresis.

For some antibody-drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, where the attachment is acysteine thiol, as in the exemplary embodiments above, an antibody mayhave only one or several cysteine thiol groups, or may have only one orseveral sufficiently reactive thiol groups through which a linker may beattached. In certain embodiments, higher drug loading, e.g. p>5, maycause aggregation, insolubility, toxicity, or loss of cellularpermeability of certain antibody-drug conjugates. In certainembodiments, the drug loading for an ADC of the invention ranges from 1to about 8; from about 2 to about 6; from about 3 to about 5; from about3 to about 4; from about 3.1 to about 3.9; from about 3.2 to about 3.8;from about 3.2 to about 3.7; from about 3.2 to about 3.6; from about 3.3to about 3.8; or from about 3.3 to about 3.7. Indeed, it has been shownthat for certain ADCs, the optimal ratio of drug moieties per antibodymay be less than 8, and may be about 2 to about 5. See US 2005-0238649A1 (herein incorporated by reference in its entirety).

In certain embodiments, fewer than the theoretical maximum of drugmoieties are conjugated to an antibody during a conjugation reaction. Anantibody may contain, for example, lysine residues that do not reactwith the drug-linker intermediate or linker reagent, as discussed below.Generally, antibodies do not contain many free and reactive cysteinethiol groups which may be linked to a drug moiety; indeed most cysteinethiol residues in antibodies exist as disulfide bridges. In certainembodiments, an antibody may be reduced with a reducing agent such asdithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partialor total reducing conditions, to generate reactive cysteine thiolgroups. In certain embodiments, an antibody is subjected to denaturingconditions to reveal reactive nucleophilic groups such as lysine orcysteine.

The loading (drug/antibody ratio) of an ADC may be controlled indifferent ways, e.g., by: (i) limiting the molar excess of drug-linkerintermediate or linker reagent relative to antibody, (ii) limiting theconjugation reaction time or temperature, (iii) partial or limitingreductive conditions for cysteine thiol modification, (iv) engineeringby recombinant techniques the amino acid sequence of the antibody suchthat the number and position of cysteine residues is modified forcontrol of the number and/or position of linker-drug attachments (suchas thioMab or thioFab prepared as disclosed herein and in WO2006/034488(herein incorporated by reference in its entirety)).

It is to be understood that where more than one nucleophilic groupreacts with a drug-linker intermediate or linker reagent followed bydrug moiety reagent, then the resulting product is a mixture of ADCcompounds with a distribution of one or more drug moieties attached toan antibody. The average number of drugs per antibody may be calculatedfrom the mixture by a dual ELISA antibody assay, which is specific forantibody and specific for the drug. Individual ADC molecules may beidentified in the mixture by mass spectroscopy and separated by HPLC,e.g. hydrophobic interaction chromatography.

In some embodiments, a homogeneous ADC with a single loading value maybe isolated from the conjugation mixture by electrophoresis orchromatography.

Methods of Determining Cytotoxic Effect of ADCs

Methods of determining whether a Drug or Antibody-Drug conjugate exertsa cytostatic and/or cytotoxic effect on a cell are known. Generally, thecytotoxic or cytostatic activity of an Antibody Drug conjugate can bemeasured by: exposing mammalian cells expressing a target protein of theAntibody Drug conjugate in a cell culture medium; culturing the cellsfor a period from about 6 hours to about 5 days; and measuring cellviability. Cell-based in vitro assays can be used to measure viability(proliferation), cytotoxicity, and induction of apoptosis (caspaseactivation) of the Antibody Drug conjugate.

For determining whether an Antibody Drug conjugate exerts a cytostaticeffect, a thymidine incorporation assay may be used. For example, cancercells expressing a target antigen at a density of 5,000 cells/well of a96-well plated can be cultured for a 72-hour period and exposed to 0.5μCi of 3H-thymidine during the final 8 hours of the 72-hour period. Theincorporation of 3H-thymidine into cells of the culture is measured inthe presence and absence of the Antibody Drug conjugate.

For determining cytotoxicity, necrosis or apoptosis (programmed celldeath) can be measured. Necrosis is typically accompanied by increasedpermeability of the plasma membrane; swelling of the cell, and ruptureof the plasma membrane. Apoptosis is typically characterized by membraneblebbing, condensation of cytoplasm, and the activation of endogenousendonucleases. Determination of any of these effects on cancer cellsindicates that an Antibody Drug conjugate is useful in the treatment ofcancers.

Cell viability can be measured by determining in a cell the uptake of adye such as neutral red, trypan blue, or ALAMAR™ blue (see, e.g., Pageet al., 1993, Intl. J. Oncology 3:473-476). In such an assay, the cellsare incubated in media containing the dye, the cells are washed, and theremaining dye, reflecting cellular uptake of the dye, is measuredspectrophotometrically. The protein-binding dye sulforhodamine B (SRB)can also be used to measure cytoxicity (Skehan et al., 1990, J. Natl.Cancer Inst. 82:1107-12).

Alternatively, a tetrazolium salt, such as MTT, is used in aquantitative colorimetric assay for mammalian cell survival andproliferation by detecting living, but not dead, cells (see, e.g.,Mosmann, 1983, J. Immunol. Methods 65:55-63).

Apoptosis can be quantitated by measuring, for example, DNAfragmentation. Commercial photometric methods for the quantitative invitro determination of DNA fragmentation are available. Examples of suchassays, including TUNEL (which detects incorporation of labelednucleotides in fragmented DNA) and ELISA-based assays, are described inBiochemica, 1999, no. 2, pp. 34-37 (Roche Molecular Biochemicals).

Apoptosis can also be determined by measuring morphological changes in acell. For example, as with necrosis, loss of plasma membrane integritycan be determined by measuring uptake of certain dyes (e.g., afluorescent dye such as, for example, acridine orange or ethidiumbromide). A method for measuring apoptotic cell number has beendescribed by Duke and Cohen, Current Protocols in Immunology (Coligan etal. eds., 1992, pp. 3.17.1-3.17.16). Cells also can be labeled with aDNA dye (e.g., acridine orange, ethidium bromide, or propidium iodide)and the cells observed for chromatin condensation and margination alongthe inner nuclear membrane. Other morphological changes that can bemeasured to determine apoptosis include, e.g., cytoplasmic condensation,increased membrane blebbing, and cellular shrinkage.

The presence of apoptotic cells can be measured in both the attached and“floating” compartments of the cultures. For example, both compartmentscan be collected by removing the supernatant, trypsinizing the attachedcells, combining the preparations following a centrifugation wash step(e.g., 10 minutes at 2000 rpm), and detecting apoptosis (e.g., bymeasuring DNA fragmentation). (See, e.g., Piazza et al., 1995, CancerResearch 55:3110-16).

In vivo, the effect of a therapeutic composition of the multispecificantibody of the invention can be evaluated in a suitable animal model.For example, xenogenic cancer models can be used, wherein cancerexplants or passaged xenograft tissues are introduced into immunecompromised animals, such as nude or SCID mice (Klein et al., 1997,Nature Medicine 3: 402-408). Efficacy can be measured using assays thatmeasure inhibition of tumor formation, tumor regression or metastasis,and the like.

The therapeutic compositions used in the practice of the foregoingmethods can be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material that when combined with the therapeutic compositionretains the anti-tumor function of the therapeutic composition and isgenerally non-reactive with the patient's immune system. Examplesinclude, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like (see, generally,Remington's Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980).

Antibody Compositions for In Vivo Administration

Formulations of the antibodies used in accordance with the presentinvention are prepared for storage by mixing an antibody having thedesired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. [1980]), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to provide antibodies with otherspecificities. Alternatively, or in addition, the composition maycomprise a cytotoxic agent, cytokine, growth inhibitory agent and/orsmall molecule antagonist. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration should besterile, or nearly so. This is readily accomplished by filtrationthrough sterile filtration membranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and .gammaethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

When encapsulated antibodies remain in the body for a long time, theymay denature or aggregate as a result of exposure to moisture at 37° C.,resulting in a loss of biological activity and possible changes inimmunogenicity. Rational strategies can be devised for stabilizationdepending on the mechanism involved. For example, if the aggregationmechanism is discovered to be intermolecular S—S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

Administrative Modalities

The antibodies and chemotherapeutic agents of the invention areadministered to a subject, in accord with known methods, such asintravenous administration as a bolus or by continuous infusion over aperiod of time, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. Intravenous or subcutaneousadministration of the antibody is preferred.

Treatment Modalities

In the methods of the invention, therapy is used to provide a positivetherapeutic response with respect to a disease or condition. By“positive therapeutic response” is intended an improvement in thedisease or condition, and/or an improvement in the symptoms associatedwith the disease or condition. For example, a positive therapeuticresponse would refer to one or more of the following improvements in thedisease: (1) a reduction in the number of neoplastic cells; (2) anincrease in neoplastic cell death; (3) inhibition of neoplastic cellsurvival; (5) inhibition (i.e., slowing to some extent, preferablyhalting) of tumor growth; (6) an increased patient survival rate; and(7) some relief from one or more symptoms associated with the disease orcondition.

Positive therapeutic responses in any given disease or condition can bedetermined by standardized response criteria specific to that disease orcondition. Tumor response can be assessed for changes in tumormorphology (i.e., overall tumor burden, tumor size, and the like) usingscreening techniques such as magnetic resonance imaging (MRI) scan,x-radiographic imaging, computed tomographic (CT) scan, bone scanimaging, endoscopy, and tumor biopsy sampling including bone marrowaspiration (BMA) and counting of tumor cells in the circulation.

In addition to these positive therapeutic responses, the subjectundergoing therapy may experience the beneficial effect of animprovement in the symptoms associated with the disease.

Thus for B cell tumors, the subject may experience a decrease in theso-called B symptoms, i.e., night sweats, fever, weight loss, and/orurticaria. For pre-malignant conditions, therapy with an multispecifictherapeutic agent may block and/or prolong the time before developmentof a related malignant condition, for example, development of multiplemyeloma in subjects suffering from monoclonal gammopathy of undeterminedsignificance (MGUS).

An improvement in the disease may be characterized as a completeresponse. By “complete response” is intended an absence of clinicallydetectable disease with normalization of any previously abnormalradiographic studies, bone marrow, and cerebrospinal fluid (CSF) orabnormal monoclonal protein in the case of myeloma.

Such a response may persist for at least 4 to 8 weeks, or sometimes 6 to8 weeks, following treatment according to the methods of the invention.Alternatively, an improvement in the disease may be categorized as beinga partial response. By “partial response” is intended at least about a50% decrease in all measurable tumor burden (i.e., the number ofmalignant cells present in the subject, or the measured bulk of tumormasses or the quantity of abnormal monoclonal protein) in the absence ofnew lesions, which may persist for 4 to 8 weeks, or 6 to 8 weeks.

Treatment according to the present invention includes a “therapeuticallyeffective amount” of the medicaments used. A “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve a desired therapeutic result.

A therapeutically effective amount may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the medicaments to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the antibody or antibody portion areoutweighed by the therapeutically beneficial effects.

A “therapeutically effective amount” for tumor therapy may also bemeasured by its ability to stabilize the progression of disease. Theability of a compound to inhibit cancer may be evaluated in an animalmodel system predictive of efficacy in human tumors.

Alternatively, this property of a composition may be evaluated byexamining the ability of the compound to inhibit cell growth or toinduce apoptosis by in vitro assays known to the skilled practitioner. Atherapeutically effective amount of a therapeutic compound may decreasetumor size, or otherwise ameliorate symptoms in a subject. One ofordinary skill in the art would be able to determine such amounts basedon such factors as the subject's size, the severity of the subject'ssymptoms, and the particular composition or route of administrationselected.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. Parenteral compositions may beformulated in dosage unit form for ease of administration and uniformityof dosage. Dosage unit form as used herein refers to physically discreteunits suited as unitary dosages for the subjects to be treated; eachunit contains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

The specification for the dosage unit forms of the present invention aredictated by and directly dependent on (a) the unique characteristics ofthe active compound and the particular therapeutic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch an active compound for the treatment of sensitivity in individuals.

The efficient dosages and the dosage regimens for the multispecificantibodies used in the present invention depend on the disease orcondition to be treated and may be determined by the persons skilled inthe art.

An exemplary, non-limiting range for a therapeutically effective amountof an multispecific antibody used in the present invention is about0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as0.3, about 1, or about 3 mg/kg. In another embodiment, the antibody isadministered in a dose of 1 mg/kg or more, such as a dose of from 1 to20 mg/kg, e.g. a dose of from 5 to 20 mg/kg, e.g. a dose of 8 mg/kg.

A medical professional having ordinary skill in the art may readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, a physician or a veterinarian couldstart doses of the medicament employed in the pharmaceutical compositionat levels lower than that required in order to achieve the desiredtherapeutic effect and gradually increase the dosage until the desiredeffect is achieved.

In one embodiment, the multispecific antibody is administered byinfusion in a weekly dosage of from 10 to 500 mg/kg such as of from 200to 400 mg/kg Such administration may be repeated, e.g., 1 to 8 times,such as 3 to 5 times. The administration may be performed by continuousinfusion over a period of from 2 to 24 hours, such as of from 2 to 12hours.

In one embodiment, the multispecific antibody is administered by slowcontinuous infusion over a long period, such as more than 24 hours, ifrequired to reduce side effects including toxicity.

In one embodiment the multispecific antibody is administered in a weeklydosage of from 250 mg to 2000 mg, such as for example 300 mg, 500 mg,700 mg, 1000 mg, 1500 mg or 2000 mg, for up to 8 times, such as from 4to 6 times. The administration may be performed by continuous infusionover a period of from 2 to 24 hours, such as of from 2 to 12 hours. Suchregimen may be repeated one or more times as necessary, for example,after 6 months or 12 months. The dosage may be determined or adjusted bymeasuring the amount of compound of the present invention in the bloodupon administration by for instance taking out a biological sample andusing anti-idiotypic antibodies which target the antigen binding regionof the multispecific antibody.

In a further embodiment, the multispecific antibody is administered onceweekly for 2 to 12 weeks, such as for 3 to 10 weeks, such as for 4 to 8weeks.

In one embodiment, the multispecific antibody is administered bymaintenance therapy, such as, e.g., once a week for a period of 6 monthsor more.

In one embodiment, the multispecific antibody is administered by aregimen including one infusion of an multispecific antibody followed byan infusion of an multispecific antibody conjugated to a radioisotope.The regimen may be repeated, e.g., 7 to 9 days later.

As non-limiting examples, treatment according to the present inventionmay be provided as a daily dosage of an antibody in an amount of about0.1-100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on atleast one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, or 40, or alternatively, at least one of week 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 afterinitiation of treatment, or any combination thereof, using single ordivided doses of every 24, 12, 8, 6, 4, or 2 hours, or any combinationthereof.

In some embodiments the multispecific antibody molecule thereof is usedin combination with one or more additional therapeutic agents, e.g. achemotherapeutic agent. Non-limiting examples of DNA damagingchemotherapeutic agents include topoisomerase I inhibitors (e.g.,irinotecan, topotecan, camptothecin and analogs or metabolites thereof,and doxorubicin); topoisomerase II inhibitors (e.g., etoposide,teniposide, and daunorubicin); alkylating agents (e.g., melphalan,chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine,semustine, streptozocin, decarbazine, methotrexate, mitomycin C, andcyclophosphamide); DNA intercalators (e.g., cisplatin, oxaliplatin, andcarboplatin); DNA intercalators and free radical generators such asbleomycin; and nucleoside mimetics (e.g., 5-fluorouracil, capecitibine,gemcitabine, fludarabine, cytarabine, mercaptopurine, thioguanine,pentostatin, and hydroxyurea).

Chemotherapeutic agents that disrupt cell replication include:paclitaxel, docetaxel, and related analogs; vincristine, vinblastin, andrelated analogs; thalidomide, lenalidomide, and related analogs (e.g.,CC-5013 and CC-4047); protein tyrosine kinase inhibitors (e.g., imatinibmesylate and gefitinib); proteasome inhibitors (e.g., bortezomib); NF-κBinhibitors, including inhibitors of IκB kinase; antibodies which bind toproteins overexpressed in cancers and thereby downregulate cellreplication (e.g., trastuzumab, rituximab, cetuximab, and bevacizumab);and other inhibitors of proteins or enzymes known to be upregulated,over-expressed or activated in cancers, the inhibition of whichdownregulates cell replication.

In some embodiments, the antibodies of the invention can be used priorto, concurrent with, or after treatment with Velcade® (bortezomib).

All cited references are herein expressly incorporated by reference intheir entirety.

Whereas particular embodiments of the invention have been describedabove for purposes of illustration, it will be appreciated by thoseskilled in the art that numerous variations of the details may be madewithout departing from the invention as described in the appendedclaims.

EXAMPLES

Examples are provided below to illustrate the present invention. Theseexamples are not meant to constrain the present invention to anyparticular application or theory of operation. For all constant regionpositions discussed in the present invention, numbering is according tothe EU index as in Kabat (Kabat et al., 1991, Sequences of Proteins ofImmunological Interest, 5th Ed., United States Public Health Service,National Institutes of Health, Bethesda, entirely incorporated byreference). Those skilled in the art of antibodies will appreciate thatthis convention consists of nonsequential numbering in specific regionsof an immunoglobulin sequence, enabling a normalized reference toconserved positions in immunoglobulin families. Accordingly, thepositions of any given immunoglobulin as defined by the EU index willnot necessarily correspond to its sequential sequence.

Example 1 Design of Non-Native Charge Substitutions to Reduce pI

Antibody constant chains were modified with lower pI by engineeringsubstitutions in the constant domains. Reduced pI can be engineered bymaking substitutions of basic amino acids (K or R) to acidic amino acids(D or E), which result in the largest decrease in pI. Mutations of basicamino acids to neutral amino acids and neutral amino acids to acidicamino acids will also result in a decrease in pI. A list of amino acidpK values can be found in Table 1 of Bjellqvist et al., 1994,Electrophoresis 15:529-539.

We chose to explore substitutions in the antibody CH1 (Cγ1) and CL(Ckappa or CK) regions (sequences are shown in FIG. 13) because, unlikethe Fc region, they do not interact with native ligands that impact theantibody's pharmacological properties. In deciding which positions tomutate, the surrounding environment and number of contacts the WT aminoacid makes with its neighbors was taken into account such as to minimizethe impact of a substitution or set of substitutions on structure and/orfunction. The solvent accessibility or fraction exposed of each CH1 andCK position was calculated using relevant crystal structures of antibodyFab domains. The results are shown in FIGS. 2 and 3 of U.S. Ser. No.13/648,951 for the Cγ1 and CK respectively (Figures and accompanyinglegends are expressly incorporated herein by reference). Design wasguided further by examining the CH1 and CL domains for positions thatare isotypic between the immunoglobulin isotypes (IgG1, IgG2, IgG3, andIgG4). Because such variations occur naturally, such positions areexpected to be amenable to substitution. Based on this analysis, anumber of substitutions were identified that reduce pI but are predictedto have minimal impact on the biophysical properties of the domains.

As for all the heterodimeric proteins herein, genes encoding the heavyand light chains of the antibodies were constructed in the mammalianexpression vector pTT5. The human IgG1 constant chain gene was obtainedfrom IMAGE clones and subcloned into the pTT5 vector. VH and VL genesencoding the anti-VEGF antibodies were synthesized commercially (BlueHeron Biotechnologies, Bothell Wash.), and subcloned into the vectorsencoding the appropriate CL and IgG1 constant chains. Amino acidmodifications were constructed using site-directed mutagenesis using theQuikChange® site-directed mutagenesis methods (Stratagene, La JollaCalif.). All DNA was sequenced to confirm the fidelity of the sequences.

Plasmids containing heavy chain gene (VH-Cγ1-Cγ2-Cγ3) wereco-transfected with plasmid containing light chain gene (VL-Cκ) into293E cells using lipofectamine (Invitrogen, Carlsbad Calif.) and grownin FreeStyle 293 media (Invitrogen, Carlsbad Calif.). After 5 days ofgrowth, the antibodies were purified from the culture supernatant byprotein A affinity using the MabSelect resin (GE Healthcare). Antibodyconcentrations were determined by bicinchoninic acid (BCA) assay(Pierce).

The pI engineered mAbs were generally characterized by SDS PAGE on anAgilent Bioanalyzer, by size exclusion chromatography (SEC), isoelectricfocusing (IEF) gel electrophoresis, binding to antigen by Biacore, anddifferential scanning calorimetry (DSC). All mAbs showed high purity onSDS-PAGE and SEC. IEF gels indicated that each variant had the designedisoelectric point. Generally the binding analysis on Biacore showed thatpI engineered variants bound to antigen with similar affinity as theparent antibodies, indicating that the designed substitutions did notperturb the function of the mAb. DSC in the Figures show which variantsgenerally had high thermostability.

Pharmacokinetic experiments for serum half life as appropriate wereperformed in B6 mice that are homozygous knock-outs for murine FcRn andheterozygous knock-ins of human FcRn (mFcRn−/−, hFcRn+) (Petkova et al.,2006, Int Immunol 18(12):1759-69, entirely incorporated by reference),herein referred to as hFcRn or hFcRn+ mice.

A single, intravenous tail vein injection of antibody (2 mg/kg) wasgiven to groups of 4-7 female mice randomized by body weight (20-30 grange). Blood (˜50 μl) was drawn from the orbital plexus at each timepoint, processed to serum, and stored at −80° C. until analysis.Antibody concentrations were determined using an ELISA assay. Serumconcentration of antibody was measured using recombinant antigen ascapture reagent, and detection was carried out with biotinylatedanti-human kappa antibody and europium-labeled streptavidin. The timeresolved fluorescence signal was collected. PK parameters weredetermined for individual mice with a non-compartmental model usingWinNonLin (Pharsight Inc, Mountain View Calif.). Nominal times and dosewere used with uniform weighing of points.

Example 2 Engineering Approaches to Constant Region pI Engineering

Reduction in the pI of a protein or antibody can be carried out using avariety of approaches. At the most basic level, residues with high pKa's(lysine, arginine, and to some extent histidine) are replaced withneutral or negative residues, and/or neutral residues are replaced withlow pKa residues (aspartic acid and glutamic acid). The particularreplacements may depend on a variety of factors, including location inthe structure, role in function, and immunogenicity.

Because immunogenicity is a concern, efforts can be made to minimize therisk that a substitution that lowers the pI will elicit immunogenicity.One way to minimize risk is to minimize the mutational load of thevariants, i.e. to reduce the pI with the fewest number of mutations.Charge swapping mutations, where a K, R, or H is replaced with a D or E,have the greatest impact on reducing pI, and so these substitutions arepreferred. Another approach to minimizing the risk of immunogenicitywhile reducing pI is to utilize substitutions from homologous humanproteins. Thus for antibody constant chains, the isotypic differencesbetween the IgG subclasses (IgG1, IgG2, IgG3, and IgG4) provide low-risksubstitutions. Because immune recognition occurs at a local sequencelevel, i.e. MHC II and T-cell receptors recognize epitopes typically 9residues in length, pI-altering substitutions may be accompanied byisotypic substitutions proximal in sequence. In this way, epitopes canbe extended to match a natural isotype. Such substitutions would thusmake up epitopes that are present in other human IgG isotypes, and thuswould be expected to be tolerized.

One approach for engineering changes in pI is to use isotype switching,as described herein.

Another approach to engineering lower pI into proteins and antibodies isto fuse negatively charged residues to the N- or C-termini. Thus forexample, peptides consisting principally of aspartic acids and glutamicacid may be fused to the N-terminus or C-terminus to the antibody heavychain, light chain or both. Because the N-termini are structurally closeto the antigen binding site, the C-termini are preferred.

Based on the described engineering approaches, a number of variants weredesigned to alter the isoelectric point of the antibody heavy chain (Fcregion generally) and in some cases the light chain.

Example 3 Isotypic Light Chain Constant Region Variants

Homology between CK and Cλ is not as high as between the IgG subclasses,however the sequence and structural homology that exists was still usedto guide substitutions to create an isotypic low-pI light chain constantregion. In FIG. 56, positions with residues contributing to a higher pI(K, R, and H) or lower pI (D and E) are highlighted in bold. Grayindicates lysine, arginines, and histidines that may be substituted,preferably with aspartic or glutamic acids, to lower the isoelectricpoint. These variants, alone or in any combination, can independentlyand optionally be combined with all other heavy chain variants inscaffolds that have at least one light chain.

Example 4 Purifying Mixtures of Antibody Variants with ModifiedIsoelectric Points

Substitutions that modify the antibody isoelectric point may beintroduced into one or more chains of an antibody variant to facilitateanalysis and purification. For instance, heterodimeric antibodies suchas those disclosed in US2011/0054151A1 can be purified by modifying theisoelectric point of one chain, so that the multiple species presentafter expression and Protein A purification can be purified by methodsthat separate proteins based on differences in charge, such as ionexchange chromatography.

As an example, the heavy chain of bevacizumab was modified byintroducing substitutions to lower its isoelectric point such that thedifference in charges between the three species produced whenWT-IgG1-HC, low-pI-HC, and WT-LC are transfected in 293E cells is largeenough to facilitate purification by anion exchange chromatography.Clones were created as described above, and transfection and initialpurification by Protein A chromatography is also as described above.Sequences of the three chains “Heavy chain 1 of XENP10653”, “Heavy chain2 of XENP10653”, and “Light chain of XENP10653” in the Figures. AfterProtein A purification, three species with nearly identical molecularweights, but different charges are obtained. These are theWT-IgG1-HC/WT-IgG1-HC homodimer (pI=8.12), WT-IgG1-HC/low-pI-HCheterodimer (pI=6.89), and low-pI-HC/low-pI-HC homodimer (pI=6.20). Themixture was loaded onto a GE HiTrap Q HP column in 20 mM Tris, pH 7.6and eluted with a step-wise gradient of NaCl consisting of 50 mM, 100mM, and finally 200 mM NaCl in the same Tris buffer. Elution wasmonitored by A280, and each fraction analyzed on Invitrogen pH 3-10 IEFgels with Novex running buffer and these results are shown in FIG. 40.WT-IgG1-HC/WT-IgG1-HC homodimer does not bind to the anion exchangecolumn at pH 7.6 and is thus present in the flowthrough and wash (lanes1-2). The desired heterodimer elutes with 50 mM NaCl (lane 3), while thelow-pI-HC/low-pI-HC homodimer binds tightest to the column and elutes at100 (lane 4) and 200 mM (lane 5) NaCl. Thus the desired heterodimervariant, which is difficult to purify by other means because of itssimilar molecular weight to the other two species, is easily purified bythe introduction of low pI substitutions into one chain. This method ofpurifying antibodies by engineering the isoelectric point of each chaincan be applied to methods of purifying various bispecific antibodyconstructs. The method is particularly useful when the desired speciesin the mixture has similar molecular weight and other properties suchthat normal purification techniques are not capable of separating thedesired species in high yield.

Example 5 Design of Non-Native Charge Substitutions to Alter pI

The pI of antibody constant chains were altered by engineeringsubstitutions in the constant domains. Reduced pI can be engineered bymaking substitutions of basic amino acids (K or R) to acidic amino acids(D or E), which result in the largest decrease in pI. Mutations of basicamino acids to neutral amino acids and neutral amino acids to acidicamino acids will also result in a decrease in pI. Conversely, increasedpI can be engineered by making substitutions of acidic amino acids (D orE) to basic amino acids (K or R), which result in the largest increasein pI. Mutations of acidic amino acids to neutral amino acids andneutral amino acids to basic amino acids will also result in a increasein pI. A list of amino acid pK values can be found in Table 1 ofBjellqvist et al., 1994, Electrophoresis 15:529-539.

In deciding which positions to mutate, the surrounding environment andnumber of contacts the WT amino acid makes with its neighbors was takeninto account such as to minimize the impact of a substitution or set ofsubstitutions on structure and/or function. The solvent accessibility orfraction exposed of each constant region position was calculated usingrelevant crystal structures. Based on this analysis, a number ofsubstitutions were identified that reduce or increase pI but arepredicted to have minimal impact on the biophysical properties of thedomains.

Calculation of protein pI was performed as follows. First, a count wastaken of the number of D, E, C, H, K, R, and Y amino acids as well asthe number of N- and C-termini present in the protein. Then, the pI wascalculated by identifying the pH for which the protein has an overallcharge of zero. This was done by calculating the net charge of theprotein at a number of test pH values. Test pH values were set in aniterative manner, stepping up from a low pH of 0 to a high pH of 14 byincrements of 0.001 until the charge of the protein reached or surpassedzero. Net charge of a protein at a given pH was calculated by thefollowing formula:

${\text{?}({pH})} = {{\text{?}\frac{\text{?}}{\text{?}}} - {\text{?}\frac{\text{?}}{\text{?}}}}$?indicates text missing or illegible when filed

where q_(protein)(pH) is the net charge on the protein at the given pH,is the number of amino acid

(or N- or C-termini) present in the protein, and is the pK of amino acid

(or N- or C-termini).

Example 6 Purifying Mixtures of Antibody Variants with ModifiedIsoelectric Points

Variants were first purified by Protein A, and then loaded onto a GEHealthcare HiTrap SP HP cation exchange column in 50 mM MES (pH 6.0) andeluted with an NaCl gradient. Following elution, fractions from eachpeak were loaded onto a Lonza IsoGel IEF plate (pH range 7-11) foranalysis. Separation of the middle pI heterodimer is achieved in eachcase, with separation improved when the heterodimer has a largerdifference in pI from the homodimers.

Example 7 Stability of pI Isosteric Variants

Differential scanning fluorimetry (DSF) was used to evaluate thestability of antibodies containing isosteric pI substitutions. DSFexperiments were performed using a Bio-Rad CFX Connect Real-Time PCRDetection System. Proteins were mixed with SYPRO Orange fluorescent dyeand diluted to 0.25 or 0.50 mg/mL in PBS. The final concentration ofSYPRO Orange was 10×. After an initial 10 minute incubation period at25° C., proteins were heated from 25 to 95° C. using a heating rate of1° C./min. A fluorescence measurement was taken every 30 sec. Meltingtemperatures were calculated using the instrument software. The resultsare shown in FIG. 44. The results indicated that isosteric(+) pIvariants had lower stability. We therefore made further variants toreduce the number of substitutions on the increased pI side, but resultsshowed that only E269Q had a small effect on stability, while E272Q andE283Q had large negative impacts on stability.

Example 8 Design of Charged scFv Linkers to Enable IEX Purification ofscFv Containing Heterodimeric Bispecific Antibodies

We have previously engineered the antibody constant regions ofheterodimeric antibodies to have higher or lower pI using both isotypicand isosteric charge substitutions. These methods enable efficient IEXpurification of heterodimeric species, but may impact stability orimmunogenicity of the antibodies due to the unnatural substitutionsintroduced. For scFv containing heterodimeric bispecific antibodies(Examples are shown in FIG. 1), another region to introduce chargedsubstitutions is the scFv linker that connects the VH and VL of scFvconstructs. The most common linker used is (GGGGS)3 or (GGGGS)4, whichhas been shown to be flexible enough to allow stable scFv formationwithout diabody formation. These sequences are already unnatural, andcontain little sequence specificity for likely immunogenic epitopes.Therefore we thought that introducing charged substitutions into scFvlinkers may be a good strategy to enable IEX purification ofheterodimeric bispecific species containing scFvs. Various positivelyand negatively charged scFv linkers were designed and are shown in 9.All linkers are novel constructs except for the “Whitlow” linker whichwas reported by Whitlow et al., (Whitlow M, Protein Eng. 1993 (8),989-995.). Linkers designated as 6paxA_(—)1 (+A) and 3hsc_(—)2 (−A) weretaken from a database of unstructured regions in human proteins obtainedfrom PDB files and these linkers are approximately the same length as(GGGGS)3 and contain positive or negative charges. Other linkers arebased on introducing repetitive Lys or Glu residues, as well as Lys-Promotifs designed to reduce the chance of proteolytic degradation in thepositively charged linkers.

Charged linkers were first evaluated for biophysical behavior in thescFv-His format and then were later constructed in anti-CD19xCD3Fab-scFv-Fc bispecific format. Genes encoding the scFv of engineeredforms of the anti-CD3 antibody SP34 or the anti-CD 19 4G7 antibody wereconstructed in the mammalian expression vector pTT5. For full-lengthconstructs, the human IgG1 constant chain gene was obtained from IMAGEclones and subcloned into the pTT5 vector. scFv genes were synthesizedcommercially (Blue Heron Biotechnologies, Bothell Wash. Amino acidmodifications were constructed using site-directed mutagenesis using theQuikChange® site-directed mutagenesis methods (Stratagene, La JollaCalif.). All DNA was sequenced to confirm the fidelity of the sequences.

Plasmids containing scFv or heavy chain and light chain genes weretransfected (or co-transfected for full-length formats) into 293E cellsusing lipofectamine (Invitrogen, Carlsbad Calif.) and grown in FreeStyle293 media (Invitrogen, Carlsbad Calif.). After 5 days of growth, theantibodies were purified from the culture supernatant by protein A(full-length) using the MabSelect resin (GE Healthcare) or using Ni-NTAresing for His-tagged scFvs. Heterodimers were further purified by ionexchange chromatography (IEX) to assess the ability of the altered pIheavy chains to enable efficient purification. Examples of IEXpurifications for an anti-CD19xCD3 bispecific containing a positivelycharged linker in the CD3 scFv is shown in FIG. 49. Antibodyconcentrations were determined by bicinchoninic acid (BCA) assay(Pierce).

The pI engineered scFvs or antibodies were characterized by SDS-PAGE,size exclusion chromatography (SEC), isoelectric focusing (IEF) gelelectrophoresis, and/or differential scanning fluorimetry (DSF).

Example 9 Stability and Behavior of scFvs Containing Charged Linkers

Anti-CD3 scFv's and anti-CD19 scFv's containing positively or negativelycharged linkers, respectively, were evaluated for SEC behavior as wellas for stability using DSF. Differential scanning fluorimetry (DSF) wasused to evaluate the stability of scFvs containing charged linkers. DSFexperiments were performed using a Bio-Rad CFX Connect Real-Time PCRDetection System. Proteins were mixed with SYPRO Orange fluorescent dyeand diluted to 0.25 or 0.50 mg/mL in PBS. The final concentration ofSYPRO Orange was 10×. After an initial 10 minute incubation period at25° C., proteins were heated from 25 to 95° C. using a heating rate of1° C./min. A fluorescence measurement was taken every 30 sec. Meltingtemperatures were calculated using the instrument software. Tm valuesfor scFvs are shown in FIG. 45. Charged linkers had only marginalimpacts on overall scFv stability as indicated by their Tm values. SECchromatograms obtained from purified scFvs are shown in FIG. 46. Highlycharged linkers have a longer elution time and noticeable peak tailsindicating that too much charge causes the scFvs to stick to the SECresin longer than expected. Binding results for positively chargedanti-CD3 scFvs binding to CD4+ T cells (FIG. 47) indicated that bindingof most scFvs was similar, with the exception of the very highly charged(GKGKS)4 scFv, which showed weaker binding. No off-target binding wasdetected when gating for CD20+ cells in PBMCs. However, when off-targetbinding was tested using SP34 cells, some amount of off-target bindingwas seen with the highest charged linkers at high concentrations (FIG.48).

Positively charged scFv linkers on the anti-CD3 scFv in an anti-CD19xCD3Fab-scFv-Fc construct had the unexpected property of reducing the amountof high molecular weight aggregation SEC chromatograms of two bispecificconstructs (13121—with standard (GGGGS)4 linker) and (13124—with chargedlinker (GKPGS)4) incubated at various concentrations confirmed thisphenomenon.

Activity of anti-CD19xCD3 constructs containing charged scFv linkers inthe anti-CD3 scFv was evaluated using an RTCC assay with PBMCs andFab-scFv-Fc format bispecific anti-CD19xCD3 antibodies containingdifferent scFv linkers (FIG. 51). Linkers have little impact on RTCCactivity, except for the highly charged linker (GKGKS)3 which has loweractivity.

Sequences for all constructs of the invention are shown in the Figures.

1. A heterodimeric antibody comprising: a) a first monomer comprising:i) a first heavy chain constant domain comprising a first variant Fcdomain; and ii) a first antigen binding domain; and b) a second monomercomprising: i) a second heavy chain constant domain comprising a secondvariant Fc domain; and ii) a second antigen binding domain; wherein oneof said first and second variant Fc domains comprise amino acidsubstitution(s) selected from the group consisting of those depicted inFIG.
 6. 2. A heterodimeric antibody according to claim 1 wherein saidfirst antigen binding domain is a scFv covalent attached to said firstheavy chain constant domain.
 3. A heterodimeric antibody according toclaim 1 wherein said heterodimeric antibody has a structure selectedfrom the structures of FIGS. 1B to 1L and 2A to 2M.
 4. A heterodimericantibody according to claim 1 wherein said first and/or second Fc domainfurther comprises amino acid substitution(s) selected from the groupconsisting of 434A, 434S, 428L, 308F, 259I, 428L/434S, 259I/308F,436I/428L, 436I or V/434S, 436V/428L, 252Y, 252Y/254T/256E,259I/308F/428L, 236A, 239D, 239E, 332E, 332D, 239D/332E, 267D, 267E,328F, 267E/328F, 236A/332E, 239D/332E/330Y, 239D, 332E/330L, 236R, 328R,236R/328R, 236N/267E, 243L, 298A and 299T.
 5. A heterodimeric antibodyaccording to claim 1 wherein one of said first and said second variantFc domains comprises the amino acid substitutions 364K/E357Q and theother of said first and said second variant Fc domains comprises theamino acid substitutions 368D/370S.
 6. A heterodimeric antibodyaccording to claim 1 wherein said first and/or second Fc domain furthercomprises amino acid substitution(s) selected from the group consistingof those listed in FIG.
 7. 7. A heterodimeric antibody according toclaim 1 wherein said first monomer comprises a heavy chain constantdomain covalently attached to an scFv and said second monomer comprisesa heavy chain and a light chain.
 8. A heterodimeric antibody accordingto a claim 1 wherein one of said monomers comprisesN208D/Q295E/N384D/Q418E/N421D.
 9. A nucleic acid composition comprisingnucleic acids encoding said first and second monomer according toclaim
 1. 10. A host cell comprising the nucleic acid composition ofclaim
 9. 11. A method of making a heterodimeric antibody according toclaim 1 comprising culturing the host cell of claim K10 under conditionswherein said heterodimeric antibody is produced and recovering saidheterodimeric antibody.
 12. A method of treating an individual in needthereof by administering a heterodimeric antibody according to claim 1.13. A heterodimeric antibody comprising: a) a first monomer comprising aheavy chain comprising: i) a first Fc domain; and ii) a single chain Fvregion (scFv) that binds a first antigen; wherein said scFv comprises acharged scFv linker; and b) a second monomer comprising: I) a firstheavy chain comprising: 1) a second Fc domain; 2) a first variable heavychain; and ii) a first light chain. 14-18. (canceled)
 19. Aheterodimeric antibody composition comprising: a) a first monomercomprising: i) a first heavy chain sequence comprising: A) a firstvariant Fc domain as compared to a human Fc domain; and B) a firstantigen-binding domain that binds to a first antigen; and ii) a secondheavy chain sequence comprising: A) a second variant Fc domain ascompared to a human Fc domain; and B) a second antigen binding domainthat binds to a second antigen; Wherein said first and second variant Fcdomains comprise a set of amino acid substitutions selected from thegroup consisting of the amino acid sets depicted in FIG.
 3. 20. Aheterodimeric antibody composition comprising: a) a first monomercomprising: i) a first heavy chain sequence comprising: A) a firstvariant Fc domain as compared to a human Fc domain; and B) a firstantigen-binding domain that binds to a first antigen; and ii) a secondheavy chain sequence comprising: A) a second variant Fc domain ascompared to a human Fc domain; and B) a second antigen binding domainthat binds to CD19 and comprises a variable heavy chain domaincomprising the amino acid sequence of H1.227 (SEQ ID NO:X) and avariable light chain selected from the group consisting of the aminoacid sequence of L1.198 (SEQ ID NO:X) and the amino acid sequence of1.199 (SEQ ID NO:X) as depicted in FIG.
 21. 21. A heterodimeric antibodycomposition comprising: a) a first monomer comprising: i) a first heavychain sequence comprising: A) a first variant Fc domain as compared to ahuman Fc domain; and B) a first antigen binding domain comprising ananti-CD3 variable region having a sequence comprising a vhCDR1 havingthe sequence T-Y-A-M-Xaa1, wherein Xaa1 is N, S or H (SEQ ID NO:435), avhCDR2 having the sequenceR-I-R-S-K-Xaa1-N-Xaa2-Y-A-T-Xaa3-Y-Y-A-Xaa4-S-V-K-G, wherein Xaa1 is Yor A, Xaa2 is N or S, Xaa3 is Y or A and Xaa4 is D or A (SEQ ID NO:436),a vhCDR3 having the sequence H-G-N-F-G-Xaa1-S-Y-V-S-W-F-Xaa2-Y, whereinXaa1 is N, D or Q and Xaa2 is A or D (SEQ ID NO:437), a vlCDR1 havingthe sequence Xaa1-S-S-T-G-A-V-T-Xaa2-Xaa3-Xaa4-Y-A-N, wherein Xaa1 is G,R or K, Xaa2 is T or S, Xaa3 is S or G and Xaa4 is N or H, (SEQ IDNO:438), a vlCDR2 having the sequence Xaa1-T-N-Xaa2-R-A-Xaa3, whereinXaa1 is G or D, Xaa2 is K or N, and Xaa3 is P or S (SEQ ID NO:439) and avlCDR3 having the sequence Xaa1-L-W-Y-S-N-Xaa2-W-V, wherein Xaa1 is A orL and Xaa2 is L or H (SEQ ID NO:440); and b) a second monomercomprising: ii) a second heavy chain sequence comprising: A) a secondvariant Fc domain as compared to a human Fc domain; and B) an anti-CD19antigen binding domain comprising a variable heavy chain domaincomprising the amino acid sequence of H1.227 (SEQ ID NO:X) and avariable light chain selected from the group consisting of the aminoacid sequence of L1.198 (SEQ ID NO:X) and the amino acid sequence of1.199 (SEQ ID NO:X) as depicted in FIG.
 21. 22-24. (canceled)
 25. Aheterodimeric antibody comprising: a) a first monomer comprising a heavychain comprising: i) a first variant Fc domain; and ii) a single chainFv region (scFv) that binds a first antigen; wherein said scFv comprisesa charged scFv linker; and b) a second monomer comprising: I) a firstheavy chain comprising: 1) a second variant Fc domain; 2) a firstvariable heavy chain; and ii) a first light chain; wherein said firstand second variant Fc domains comprise amino acid substitution(s)selected from the group consisting of those depicted in FIG.
 7. 26. Aheterodimeric antibody composition comprising: a) a first monomercomprising: i) a first antigen binding domain comprising an anti-CD3variable region having a sequence comprising a vhCDR1 having thesequence T-Y-A-M-Xaa1, wherein Xaa1 is N, S or H (SEQ ID NO:435), avhCDR2 having the sequenceR-I-R-S-K-Xaa1-N-Xaa2-Y-A-T-Xaa3-Y-Y-A-Xaa4-S-V-K-G, wherein Xaa1 is Yor A, Xaa2 is N or S, Xaa3 is Y or A and Xaa4 is D or A (SEQ ID NO:436),a vhCDR3 having the sequence H-G-N-F-G-Xaa1-S-Y-V-S-W-F-Xaa2-Y, whereinXaa1 is N, D or Q and Xaa2 is A or D (SEQ ID NO:437), a vlCDR1 havingthe sequence Xaa1-S-S-T-G-A-V-T-Xaa2-Xaa3-Xaa4-Y-A-N, wherein Xaa1 is G,R or K, Xaa2 is T or S, Xaa3 is S or G and Xaa4 is N or H, (SEQ IDNO:438), a vlCDR2 having the sequence Xaa1-T-N-Xaa2-R-A-Xaa3, whereinXaa1 is G or D, Xaa2 is K or N, and Xaa3 is P or S (SEQ ID NO:439) and avlCDR3 having the sequence Xaa1-L-W-Y-S-N-Xaa2-W-V, wherein Xaa1 is A orL and Xaa2 is L or H (SEQ ID NO:440); ii) a first heavy chain sequencecomprising a first variant Fc domain as compared to a human Fc domain;and b) a second monomer comprising: i) a second antigen-binding domain;and ii) a second heavy chain sequence comprising a second variant Fcdomain as compared to a human Fc domain; wherein said first and secondvariant Fc domains have different amino acid sequences. 27-54.(canceled)
 55. A heterodimeric antibody comprising: a) a first heavychain comprising: i) a first Fc domain; and ii) a single chain Fv region(scFv) that binds CD3 comprising aa vhCDR1 having the sequenceT-Y-A-M-Xaa1, wherein Xaa1 is N, S or H (SEQ ID NO:435), a vhCDR2 havingthe sequence R-I-R-S-K-Xaa1-N-Xaa2-Y-A-T-Xaa3-Y-Y-A-Xaa4-S-V-K-G,wherein Xaa1 is Y or A, Xaa2 is N or S, Xaa3 is Y or A and Xaa4 is D orA (SEQ ID NO:436), a vhCDR3 having the sequenceH-G-N-F-G-Xaa1-S-Y-V-S-W-F-Xaa2-Y, wherein Xaa1 is N, D or Q and Xaa2 isA or D (SEQ ID NO:437), a vlCDR1 having the sequenceXaa1-S-S-T-G-A-V-T-Xaa2-Xaa3-Xaa4-Y-A-N, wherein Xaa1 is G, R or K, Xaa2is T or S, Xaa3 is S or G and Xaa4 is N or H, (SEQ ID NO:438), a vlCDR2having the sequence Xaa1-T-N-Xaa2-R-A-Xaa3, wherein Xaa1 is G or D, Xaa2is K or N, and Xaa3 is P or S (SEQ ID NO:439) and a vlCDR3 having thesequence Xaa1-L-W-Y-S-N-Xaa2-W-V, wherein Xaa1 is A or L and Xaa2 is Lor H (SEQ ID NO:440); and b) a second heavy chain comprising: i) asecond Fc domain; ii) a first variable heavy chain; and ii) a firstvariable light chain; wherein said first and second Fc domains aredifferent.
 56. A method of making a heterodimeric antibody comprising:a) providing a first nucleic acid encoding a first heavy chaincomprising: i) a first heavy chain comprising: 1a first Fc domain; and 2a single chain Fv region (scFv) that binds a first antigen; wherein saidscFv comprises a charged linker; and b) providing a second nucleic acidencoding a second heavy chain comprising: i) a second Fc domain; ii) afirst variable heavy chain; and c) providing a third nucleic acidcomprising a light chain; d) expressing said first, second and thirdnucleic acids in a host cell to produce a first, second and third aminoacid sequence, respectively; e) loading said first, second and thirdamino acid sequences onto an ion exchange column; and f) collecting theheterodimeric fraction.
 57. A composition comprising a heterodimerprotein comprising: a) a first monomer comprising: i) a first variantheavy chain constant region; ii) a first fusion partner; and b) a secondmonomer comprising: i) a second variant heavy chain constant region; ii)a second fusion partner; wherein the pIs of said first and secondvariant heavy chain constant regions are at least 0.5 logs apart.
 58. Acomposition comprising a heterodimer protein comprising: a) a firstmonomer comprising: i) a first variant heavy chain constant region; ii)a first fusion partner; and b) a second monomer comprising: i) a secondvariant heavy chain constant region; ii) a second fusion partner;wherein the Fc region of said first and second constant regions comprisea set of amino acid substitutions from FIGS. 3 and
 12. 59-74. (canceled)